JP2022539453A - Antigen-specific artificial immunoregulatory T (airT) cells - Google Patents

Abstract

Some of the embodiments of the compositions and methods disclosed herein comprise gene-edited artificial immune regulatory T cells (airT cells), which are natural regulatory T cells (Treg cells). or suppressor T cells) and the introduced T cell receptor (TCR) (e.g., gene editing, viral vector transduction, transfection or other including TCRs artificially produced by genetic engineering methodology). In some embodiments, the TCR is preferably specific for antigens associated with autoimmune, allergic or other inflammatory diseases. Some embodiments comprise methods of making airT cells of the invention and/or uses of airT cells of the invention. Some of such embodiments are for the treatment and/or alleviation of diseases in which antigen-specific immunosuppression may be beneficial (e.g., autoimmune diseases, allergic diseases, or other inflammatory diseases). , including the use of airT cells of the invention.

Description

Cross-reference to related applications No. 62/867,670, entitled "Antigen-Specific Treg Therapy for Autoimmune Diseases," filed May 27, 2003, which applications are incorporated herein by reference in their entirety. explicitly incorporated in the book.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with government support under contract number U01AI101981 awarded by the National Institutes of Health (NIH) and government support under contract number W81XWH-15-1-0003 awarded by the Department of Defense. It was made with support. The United States Government has certain rights in this invention.

REFERENCE TO SEQUENCE LISTING This application has been filed with the Sequence Listing in electronic form. This sequence listing is provided as a file of approximately 550 kb created on June 23, 2020 under the file name SCRI252WOSEQLIST. The information contained in this electronic form of the Sequence Listing is hereby incorporated by reference in its entirety.

Some of the embodiments provided herein comprise antigen-specific artificial immunoregulatory T (airT) cells. AirT cells of the present invention stably reprogrammed to exhibit specific regulatory T cell (Treg) characteristics by gene editing, and by gene editing, viral vector transduction, transfection or other genetic engineering methodologies , artificial immune system T lymphocytes engineered to express a desired functional T cell antigen receptor (TCR) or other antigen receptor (such as a chimeric antigen receptor (CAR)). In some embodiments, the airT cells of the invention are capable of exerting immunosuppressive activity in response to recognition of specific antigens by their TCR.

Autoimmune diseases such as type 1 diabetes, multiple sclerosis, myocarditis, rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE) (also called 'lupus') are chronic diseases with altered immunological self-tolerance As a result, immune activation abnormalities and end-organ pathologies occur, which are often life-threatening. Inappropriate and deleterious dysregulation of immune tolerance may also contribute to pathologies associated with allergy, asthma, transplant rejection and/or graft-versus-host disease (GVHD). Thymus-derived antigen-recognizing T lymphocytes with specific functions known as regulatory T cells (Treg) (also called suppressor T cells) play a role in maintaining immune tolerance and preventing autoimmunity. It has been well documented that various autoimmune diseases are characterized by dysfunction or dysregulation of the Treg compartment.

Adoptive Transfer of Selected Functional Treg Cells Based on Their Immunosuppressive Potential to Subjects with Autoimmune Diseases as a Potential Treatment for Autoimmune Diseases is Investigated in Mouse Models and Early Clinical Trials It is However, such Treg cells lack specificity for self-antigens and exhibit unregulated cellular plasticity (e.g., from immunosuppressive negative regulators to proinflammatory effectors in the immune system). phenotype), resulting in loss of immunosuppressive Treg activity. These two major limitations preclude adoptive transfer therapy of Treg cells from yielding effective and lasting therapeutic benefits. The use of immunosuppressive Treg cells selected to respond antigen-specifically to disease-associated autoantigens may be a safer and more effective adoptive transfer therapy than simple transfer of multispecific Treg cells. ing. In this regard, the autoantigen-specific Treg cells, when infused into a subject, are expected to be specifically recruited to tissue sites where autoimmune activity is expressed, and importantly, autoimmune diseases. It is thought to exert immunosuppression specifically in response to the autoantigens responsible for the pathogenesis of . In support of this notion, studies using mouse models of autoimmune disease have shown that antigen-specific Treg cells are more effective than polyclonal Treg cells (Duggleby et al., 2018 Front. Immunol. 9:252; Tang et al 2004 J Exp Med. 199(11):1455-1465; Tarbell et al 2004 J Exp Med 199:1467-1477.).

However, the therapeutic use of adoptively transferred antigen-specific Treg cells for the treatment of autoimmune diseases, and even polyclonal Treg cells for the same use, is limited by various problems, particularly their rarity. It is difficult to isolate sufficient amounts of antigen-specific Treg cells from natural sources such as blood and lymph, and there are only a few natural Treg cells in the whole peripheral blood. The problem is that only about 1-4% of blood mononuclear cells are present. Furthermore, there is also the problem that it is difficult to expand the Treg cell population to a number suitable for therapeutic use ex vivo while maintaining immunosuppressive function. The development of Treg adoptive transfer therapy is also complicated by the fact that low-potency Treg cells survive and proliferate in the human body. Yet another issue is the plasticity of Treg cells, such as their ability to switch from an immunosuppressive negative regulator to a proinflammatory effector-like phenotype in the setting of inflammation in vivo ( 5:Art. 46; Trzonkowski et al., 2015 Sci. Translat. Med. 7(304):ps18 Romano et al., 2016 Transplant. al., 2017 Front. Immunol. 8: Art. 1517).

Prior art methods provide desired immunosuppression, such as specificity for antigens involved in the etiology of diseases for which antigen-specific immunosuppression would be beneficial (e.g., autoimmune diseases, allergies and/or other inflammatory diseases). Large populations of stable Treg cells with antigen specificity and suppressive activity have not been provided.

Therefore, in vitro and in vivo, antigen-specific immunosuppressive properties can be maintained without exhibiting plasticity, and are useful for administration to subjects in need of antigen-specific immunosuppression by adoptive transfer immunotherapy, There remains a need for stable antigen-specific immunoregulatory cells. The embodiments provided herein address these needs and provide related advantages beyond those discussed above.

Some of the embodiments of the methods and compositions provided herein are antigen-specific CD4+CD25+ artificial immunoregulatory T (airT) cells comprising:
(a) an artificially modified FOXP3 gene that constitutively expresses the forkhead box protein 3/winged helix transcription factor (FOXP3) gene product at an expression level equal to or greater than that of natural regulatory T (Treg) cells; and (b) airT cells comprising at least one introduced polynucleotide encoding an antigen-specific T cell receptor (TCR) polypeptide.

In some embodiments, artificial modification of the forkhead box protein 3/winged helix transcription factor (FOXP3) gene in CD4+CD25- T cells to encode an antigen-specific T cell receptor (TCR) polypeptide An antigen-specific CD4+CD25+ artificial immunoregulatory T (airT) cell obtained by introducing at least one polynucleotide that
The artificial modification provides airT cells that constitutively express the FOXP3 gene product at an expression level equal to or higher than that of natural regulatory T (Treg) cells.

In some embodiments, the FOXP3 gene is at a FOXP3 locus comprising an intronic T regulatory cell (Treg)-specific demethylation region (TSDR) having multiple cytosine-guanine (CG) dinucleotides, Each CG dinucleotide contains a methylated cytosine (C) nucleotide at a nucleotide position that would contain a demethylated C nucleotide in a native Treg cell. In some embodiments, at least 80%, 85%, 90%, 95%, 96%, 97%, 98% of the C nucleotides at nucleotide positions that include demethylated C nucleotides in the TSDR of natural Treg cells % or 99% are methylated. In some embodiments, said FOXP3 gene product is expressed at a level sufficient for said airT cells to maintain a CD4+CD25+ phenotype in vitro for at least 21 days. In some embodiments, the FOXP3 gene product is mediated by the airT cells in vivo following adoptive transfer of the airT cells into an immunocompetent mammalian host in need of antigen-specific immunosuppression. Expressed at levels sufficient to maintain the phenotype for at least 60 days. In some embodiments, said airT cells comprise a phenotype selected from (i) HeliosLo, (ii) CD152+, (iii) CD127- and (iv) ICOS+. In some embodiments, the artificial modification comprises knocking out the native FOXP3 locus.

In some embodiments, the artificial modification comprises insertion into the native FOXP3 locus of a nucleic acid molecule comprising a constitutively active promoter, which encodes endogenous FOXP3 at the FOXP3 locus. placed in the FOXP3 gene so as to be able to promote transcription of a nucleotide sequence that In some embodiments, the inserted nucleic acid molecule further comprises a nucleic acid sequence encoding a first CISC component capable of specifically binding to a chemically induced signaling complex (CISC) inducible molecule. . In some embodiments, the introduced polynucleotide encoding said TCR polypeptide is a second CISC construct, separate from the first CISC component, capable of specifically binding to said CISC-inducing molecule. Further includes nucleic acid sequences that encode elements. In some embodiments, the nucleic acid sequence encoding the first CISC component and the nucleic acid sequence encoding the second CISC component are separate from the first CISC component and the second CISC component. further comprising a nucleic acid sequence encoding a third CISC component capable of specifically binding to said CISC-inducing molecule. In some embodiments, the nucleic acid molecule comprising the constitutively active promoter is inserted downstream of an intronic regulatory T cell (Treg)-specific demethylation region (TSDR) of the native FOXP3 locus. ing. In some embodiments, said constitutively active promoter is the MND promoter. In some embodiments, the artificial modification is a nucleic acid molecule comprising a polynucleotide encoding exogenous FOXP3 and a constitutively active promoter operably linked to the native FOXP3 locus. including the insertion of In some embodiments, the inserted nucleic acid molecule comprising a polynucleotide encoding exogenous FOXP3 and a constitutively active promoter operably linked thereto is a chemically-inducible signaling complex (CISC ) further comprising a nucleic acid sequence encoding a first CISC component capable of specifically binding to the inducer molecule. In some embodiments, the introduced polynucleotide encoding said TCR polypeptide is a second CISC construct, separate from the first CISC component, capable of specifically binding to said CISC-inducing molecule. Further includes nucleic acid sequences that encode elements. In some embodiments, at least one of said nucleic acid sequence encoding a first CISC component and said nucleic acid sequence encoding a second CISC component is further comprising a nucleic acid sequence encoding a third CISC component capable of specifically binding said CISC-inducing molecule, separate from said CISC-inducing molecule.

In some embodiments, the nucleic acid molecule comprising a polynucleotide encoding exogenous FOXP3 and a constitutively active promoter operably linked thereto comprises an intronic regulatory T of the native FOXP3 locus. It is inserted downstream of the cell (Treg)-specific demethylation region (TSDR). In some embodiments, said constitutively active promoter is the MND promoter.

In some embodiments, the artificial modification comprises an exogenous FOXP3-encoding polynucleotide and a constitutively active promoter operably linked thereto to a chromosomal site other than the native FOXP3 locus. including insertion of a nucleic acid molecule comprising In some embodiments, at least one native T cell receptor (TCR) locus of said airT cell is knocked out or inactivated, encoding an antigen-specific T cell receptor (TCR) polypeptide. replaced by at least one introduced polynucleotide. In some embodiments, the at least one native TCR locus that is knocked out or inactivated is the native TCR α chain (TRAC) locus. In some embodiments, the inserted nucleic acid molecule comprising a polynucleotide encoding exogenous FOXP3 and a constitutively active promoter operably linked thereto is a chemically-inducible signaling complex (CISC ) further comprising a nucleic acid sequence encoding a first CISC component capable of specifically binding to the inducer molecule. In some embodiments, the introduced polynucleotide encoding said TCR polypeptide is a second CISC construct, separate from the first CISC component, capable of specifically binding to said CISC-inducing molecule. Further includes nucleic acid sequences that encode elements. In some embodiments, at least one of said nucleic acid sequence encoding a first CISC component and said nucleic acid sequence encoding a second CISC component is further comprising a nucleic acid sequence encoding a third CISC component capable of specifically binding said CISC-inducing molecule, separate from said CISC-inducing molecule. In some embodiments, said constitutively active promoter is the MND promoter. In some embodiments, said chromosome, other than the native FOXP3 locus, into which said nucleic acid molecule comprising an exogenous FOXP3-encoding polynucleotide and a constitutively active promoter operably linked thereto has been inserted. The site is within the T cell receptor alpha chain (TRAC) locus.

In some embodiments, at least one native T cell receptor (TCR) locus of said airT cell is knocked out or inactivated, encoding an antigen-specific T cell receptor (TCR) polypeptide. replaced by at least one introduced polynucleotide. In some embodiments, the at least one native TCR locus that is knocked out is the native TCR α chain (TRAC) locus.

In some embodiments, an antigen-specific CD4+CD25+ artificial immunoregulatory T (airT) cell comprising:
(a) an introduced nucleic acid encoding an exogenous forkhead box protein 3/winged helix transcription factor (FOXP3) gene product that is constitutively expressed at levels equal to or greater than those of natural regulatory T (Treg) cells; and (b) at least one introduced polynucleotide encoding an antigen-specific exogenous T-cell receptor (TCR) polypeptide,
The introduced nucleic acid sequence encoding the exogenous FOXP3 gene product comprises a nucleic acid sequence encoding a first CISC component capable of specifically binding to a chemically-inducible signaling complex (CISC)-inducible molecule. further includes;
A nucleic acid in which the introduced nucleic acid sequence encoding the exogenous TCR gene product encodes a second CISC component, separate from the first CISC component, capable of specifically binding to the CISC-inducing molecule. further containing an array,
Provide airT cells.

In some embodiments, an antigen-specific CD4+CD25+ artificial immunoregulatory T (airT) cell comprising:
(a) at the native FOXP3 locus that has been knocked out or inactivated by homologous recombination repair, (i) a constitutively active agent capable of promoting transcription of the endogenous FOXP3-encoding nucleotide sequence of the native FOXP3 gene; or (ii) a nucleotide sequence encoding an exogenous FOXP3 protein or functional derivative thereof and a constitutively active promoter operably linked thereto. a FOXP3 locus that constitutively expresses the FOXP3 gene product at levels equal to or greater than those of natural regulatory T (Treg) cells; and (b) antigen-specific exogenous T cells knocked out by homologous recombination repair. comprising a native T cell receptor alpha chain (TRAC) locus into which at least one introduced polynucleotide encoding a receptor (TCR) polypeptide has been inserted;
said inserted nucleic acid molecule comprising said constitutively active promoter, or said comprising a nucleotide sequence encoding an exogenous FOXP3 protein or a functional derivative thereof and a constitutively active promoter operably linked thereto; the inserted nucleic acid molecule further comprising a nucleic acid sequence encoding a first CISC component capable of specifically binding to a chemically-inducible signaling complex (CISC)-inducible molecule;
A nucleic acid in which the introduced nucleic acid sequence encoding said exogenous TCR polypeptide encodes a second CISC component, separate from the first CISC component, capable of specifically binding to said CISC-derived molecule further containing an array,
Provide airT cells.

In some embodiments, at least one of said nucleic acid sequence encoding a first CISC component and said nucleic acid sequence encoding a second CISC component is further comprising a nucleic acid sequence encoding a third CISC component capable of specifically binding said CISC-inducing molecule, separate from said CISC-inducing molecule. In some embodiments, said airT cells comprise at least an introduced first polynucleotide encoding an antigen-specific TCR polypeptide and an introduced second polynucleotide encoding an antigen-specific TCR polypeptide. , the first polynucleotide encodes a TCR Vα polypeptide, the second polynucleotide encodes a TCR Vβ polypeptide, and the Vα polypeptide and the Vβ polypeptide specifically recognize an antigen constitute a functional TCR that can be In some embodiments, said airT cells express an antigen-specific T cell receptor (TCR) comprising an antigen-specific TCR polypeptide encoded by at least one introduced polynucleotide encoding said TCR polypeptide. Immunosuppression can be induced in an antigen-specific manner in response to HLA-restricted stimulation by an antigen that is expressed and specifically recognized by the TCR polypeptide.
In some embodiments, said immunosuppression induced in an antigen-specific manner comprises
(i) activation and/or proliferation of effector T cells that recognize an antigen specifically recognized by said airT cell TCR comprising said TCR polypeptide encoded by said at least one introduced polynucleotide; Suppression,
(ii) expression of an inflammatory cytokine or inflammatory mediator by effector T cells that recognize an antigen specifically recognized by said airT cell TCR comprising said TCR polypeptide encoded by said at least one introduced polynucleotide; suppression of
(iii) production of one or more immunosuppressive cytokines, perforin/granzymes or anti-inflammatory products by said airT cells, or against indoleamine-2,3-dioxygenase (IDO), IL2 or adenosine in said airT cells induction of at least one of competition, tryptophan catabolism, and expression of an inhibitory receptor; and (iv) specificity by said airT cell TCR comprising said TCR polypeptide encoded by said at least one introduced polynucleotide. suppression of one or both of activation and proliferation of effector T cells that do not recognize the antigen that is systematically recognized.

In some embodiments, the TCR specifically recognizes antigens associated with the pathogenesis of autoimmune, allergic or inflammatory diseases.
In some embodiments,
(i) the autoimmune disease is type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, Crohn's disease, bullous pemphigoid, pemphigus vulgaris, autoimmune hepatitis, psoriasis , Sjögren's syndrome and celiac disease;
(ii) said allergic disease is selected from allergic asthma, pollen allergy, food allergy, drug hypersensitivity and contact dermatitis;
(iii) said inflammatory disease is selected from islet cell transplantation, asthma, hepatitis, inflammatory bowel disease (IBD), ulcerative colitis, graft versus host disease (GVHD), transplant tolerance induction, transplant rejection and sepsis be done.
In some embodiments,
(i) the antigen associated with the pathogenesis of said autoimmune disease is selected from the autoantigens described in any of Figures 141-144;
(ii) the antigen associated with the etiology of the allergic disease is selected from allergic antigens according to any one of Figures 141-144;
(iii) the antigen associated with the pathogenesis of said inflammatory disease is selected from the inflammation-associated antigens described in any of Figures 141-144;

In some embodiments, the airT cell has no more than 35, no more than 34, no more than 33, no more than 32 amino acid sequences selected from the antigenic polypeptide sequences set forth in any of Figures 141-144, 31 or less, 30 or less, 29 or less, 28 or less, 27 or less, 26 or less, 25 or less, 24 or less, 23 or less, 22 or less, 21 or less, 20 or less, 19 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, or 7 or less at least one introduced polynucleotide sequence encoding a TCR polypeptide that specifically binds to a human HLA-restricted antigenic polypeptide epitope consisting of contiguous amino acids, or a nucleotide sequence according to any of Figures 139-140 at least one introduced polynucleotide sequence encoding a TCR polypeptide encoded by In some embodiments, the airT cell has no more than 35, no more than 34, no more than 33, no more than 32 amino acid sequences selected from the antigenic polypeptide sequences set forth in any of Figures 141-144, 31 or less, 30 or less, 29 or less, 28 or less, 27 or less, 26 or less, 25 or less, 24 or less, 23 or less, 22 or less, 21 or less, 20 or less, 19 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, or 7 or less an introduced first polynucleotide sequence encoding a TCR Vα polypeptide of a TCR that specifically binds to an antigenic polypeptide epitope consisting of contiguous amino acids in a human HLA-restricted manner and encoding a TCR Vβ polypeptide of said TCR; an introduced second polynucleotide sequence; or an introduced first polynucleotide sequence encoding a TCR Vα polypeptide of a TCR comprising any of the TCRα polypeptide sequences set forth in any of Figures 136-140 and said an introduced second polynucleotide sequence encoding a TCR TCR Vβ polypeptide; or an introduced first polynucleotide sequence encoding a TCR TCR Vα polypeptide encoded by a nucleotide sequence set forth in any of FIGS. and an introduced second polynucleotide sequence encoding a TCR Vβ polypeptide of said TCR. In some embodiments, the airT cell comprises an introduced first polynucleotide sequence encoding a TCR Vα polypeptide of a TCR that specifically binds to an antigenic polypeptide in a human HLA-restricted manner and a TCR of the TCR The TCR Vα and Vβ polypeptides comprise at least an introduced second polynucleotide sequence encoding a Vβ polypeptide, wherein the TCR Vα and Vβ polypeptides are the paired TCR Vα and TCR Vβ polypeptide sequences set forth in FIG. Contains paired sequences selected from sequences.

In some embodiments, biological Treg activity induced in said airT cells in response to MHC-restricted stimulation by an antigen recognized by a TCR polypeptide encoded by said at least one introduced polynucleotide. is enhanced over the biological Treg activity of the airT cells as controls without MHC-restricted stimulation with the same antigen,
Said biological Treg activity is
(i) activation and/or proliferation of effector T cells that recognize an antigen specifically recognized by said airT cell TCR comprising said TCR polypeptide encoded by said at least one introduced polynucleotide; Suppression,
(ii) expression of an inflammatory cytokine or inflammatory mediator by effector T cells that recognize an antigen specifically recognized by said airT cell TCR comprising said TCR polypeptide encoded by said at least one introduced polynucleotide; suppression of
(iii) production of one or more immunosuppressive cytokines, perforin/granzymes or anti-inflammatory products by said airT cells, or against indoleamine-2,3-dioxygenase (IDO), IL2 or adenosine in said airT cells induction of at least one of competition, tryptophan catabolism, and expression of an inhibitory receptor; and (iv) specificity by said airT cell TCR comprising said TCR polypeptide encoded by said at least one introduced polynucleotide. suppression of one or both of activation and proliferation of effector T cells that do not recognize the antigen that is systematically recognized.

In some embodiments,
(1) the antigen associated with the pathogenesis of an autoimmune disease is an IGRP peptide (residues 241-270), the autoimmune disease is type 1 diabetes, and the TCR is HLA DRB1*0404-restricted or (2) said antigen associated with the pathogenesis of autoimmune diseases is the IGRP peptide (residues 305-324). , the autoimmune disease is type 1 diabetes, and the TCR is TCR T1D5, which recognizes the IGRP peptide (residues 305-324) in an HLA DRB1*0404-restricted manner.

Some of the embodiments of the methods and compositions provided herein are any of the airT cells for use in treating, inhibiting or alleviating autoimmune, allergic or inflammatory diseases, wherein said autoimmune disease is selected from type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, Crohn's disease, bullous pemphigoid, pemphigus vulgaris and autoimmune hepatitis immunological disease, etc., wherein the allergic disease is selected from allergic asthma, pollen allergy, food allergy, drug hypersensitivity, and contact dermatitis, and the inflammatory disease is pancreatic islet cell transplantation, asthma, Including airT cells, such as inflammatory diseases selected from hepatitis, inflammatory bowel disease (IBD), ulcerative colitis, graft versus host disease (GVHD), transplant tolerance induction, transplant rejection and sepsis.

In some embodiments, the TCR polypeptide binds an antigen associated with a disorder selected from type 1 diabetes, multiple sclerosis, myocarditis, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE).

In some embodiments, the antigen is selected from the group consisting of vimentin, aggrecan, cartilage intermediate layer protein (CILP), preproinsulin, IGRP (islet-specific glucose-6-phosphatase catalytic subunit-related protein) and enolase. be.

In some embodiments, said antigen comprises an epitope selected from the group consisting of Enol326, CILP297-1, Vim418, Agg520 and SLE3.

In some embodiments, the antigen comprises an epitope having an amino acid sequence set forth in any of SEQ ID NOs: 1363-1376 and 1408-1415.

In some embodiments, the TCR polypeptide has a CD3α polypeptide having an amino acid sequence set forth in any of SEQ ID NOs:1377-1390; and/or an amino acid sequence set forth in any of SEQ ID NOs:1377-1390 It contains a CD3β polypeptide.

Some of the embodiments of the methods and compositions provided herein include pharmaceutical compositions comprising any of the airT cells described above and a pharmaceutically acceptable excipient.

Some of the embodiments of the methods and compositions provided herein comprise the use of any of the aforementioned airT cells as a medicament.

In some embodiments, a method of producing an antigen-specific artificial immunoregulatory T (airT) cell comprising:
(a) under conditions and for a time sufficient to knock out or inactivate the native FOXP3 locus of CD4+ T cells and insert all or part of the FOXP3 locus donor template nucleic acid,
(1) a FOXP3 guide RNA (gRNA) comprising a spacer sequence complementary to a sequence within the native forkhead box protein 3/winged helix transcription factor (FOXP3) gene of CD4+ T cells, or encoding said FOXP3 gRNA; nucleic acids;
(2) a DNA endonuclease capable of forming a complex with the FOXP3 gRNA of (1), or a nucleic acid encoding said DNA endonuclease; and (3) (i) a nucleotide encoding endogenous FOXP3 of said FOXP3 gene. A nucleic acid molecule comprising a constitutively active promoter capable of promoting transcription of a sequence and (ii) a nucleotide sequence encoding a FOXP3 protein or functional derivative thereof and a constitutively active promoter operably linked thereto. and (b) simultaneously with (a) or sequentially with (a) in any order, an antigen-specific A method is provided comprising transducing said CD4+ T cells with at least one polynucleotide encoding a T cell receptor (TCR) polypeptide.
In some embodiments, step (b) comprises:
(i) transducing said CD4+ T cells with at least one retroviral vector comprising a polynucleotide encoding said antigen-specific T cell receptor (TCR) polypeptide; and (ii) said CD4+ T cells. under conditions and for a time sufficient to knock out or inactivate the native T-cell receptor alpha chain (TRAC) locus of the
(1) a TRAC guide RNA (gRNA) comprising a spacer sequence complementary to a sequence within the natural TRAC locus of said CD4+ T cell, or a nucleic acid encoding said TRAC gRNA;
(2) a DNA endonuclease capable of forming a complex with the TRAC gRNA of (1), or a nucleic acid encoding the DNA endonuclease; and (3) the antigen-specific T-cell receptor (TCR) polypeptide introducing into said CD4+ T cells a TRAC locus donor template comprising at least one encoding polynucleotide.

In some embodiments, a method of producing an antigen-specific artificial immunoregulatory T (airT) cell comprising:
(a) conditions sufficient to knock out the native T cell receptor alpha chain (TRAC) locus of CD4+ T cells and insert all or part of the first TRAC locus donor template nucleic acid and in time,
(1) a first TRAC guide RNA (gRNA) comprising a first spacer sequence complementary to a first sequence in the native TRAC locus of CD4+ T cells, or encoding said first TRAC gRNA; nucleic acids;
(2) a first DNA endonuclease capable of forming a complex with the first TRAC gRNA of (1), or a nucleic acid encoding said first DNA endonuclease; and (3) (i) FOXP3 protein or a nucleic acid molecule comprising a nucleotide sequence encoding a functional derivative thereof and (ii) a nucleic acid molecule comprising a nucleotide sequence encoding a FOXP3 protein or a functional derivative thereof and a constitutively active promoter operably linked thereto. and (b) simultaneously with (a) or sequentially with (a) in any order, said CD4+ T cells under conditions and for a time sufficient to knock out or inactivate the native T-cell receptor alpha chain (TRAC) locus of the second TRAC locus donor template nucleic acid in whole or in part,
(1) a second TRAC guide RNA (gRNA) comprising a second spacer sequence complementary to a second sequence in the TRAC gene, different from the first spacer sequence, or the second TRAC gRNA; encoding nucleic acid;
(2) selected from the same DNA endonuclease as the first DNA endonuclease and a DNA endonuclease different from the first DNA endonuclease, capable of forming a complex with the second TRAC gRNA of (1); or a nucleic acid encoding said second DNA endonuclease; and (3) at least one polynucleotide encoding an antigen-specific T-cell receptor (TCR) polypeptide. A method is provided comprising introducing a TRAC locus donor template into said CD4+ T cells.

In some embodiments, a method of producing an antigen-specific artificial immunoregulatory T (airT) cell comprising:
Homologous recombination repair knocks out or inactivates the native T-cell receptor alpha chain (TRAC) locus of CD4+ T cells, sufficient to insert all or part of the TRAC locus donor template. in terms and time,
(1) a TRAC guide RNA (gRNA) comprising a spacer sequence complementary to a sequence within the natural TRAC locus of CD4+ T cells, or a nucleic acid encoding said TRAC gRNA;
(2) a DNA endonuclease capable of forming a complex with the TRAC gRNA of (1), or a nucleic acid encoding said DNA endonuclease; and (3) encoding an antigen-specific T-cell receptor (TCR) polypeptide. A method is provided comprising introducing into a CD4+ T cell a TRAC locus donor template comprising at least one polynucleotide that does.
In some embodiments, one of said inserted donor templates (first donor template) is a first CISC construct capable of specifically binding a chemically-inducible signaling complex (CISC)-inducing molecule. further comprising a nucleic acid sequence encoding the element;
The other of said inserted donor templates (second donor template) encodes a second CISC component, separate from the first CISC component, capable of specifically binding to said CISC-inducing molecule. further includes nucleic acid sequences that
In some embodiments, at least one of the inserted first donor template and second donor template is directed to the CISC-derived molecule, separate from the first CISC component and the second CISC component. Further includes a nucleic acid sequence encoding a third CISC component capable of specific binding.
In some embodiments,
(a) said DNA endonuclease is selected from CRISPR/Cas, TALENs, meganucleases, megaTALs and zinc finger nucleases;
(b) said constitutively active promoter is MND, said insertion is by a mechanism selected from homologous recombination repair and non-homologous end joining;
(d) the first CISC component and the second CISC component are mutually exclusive selected from IL2RB and IL2RG;
(e) the third CISC component is FKBP;
(f) said CISC-derived molecule is rapamycin or an analogue thereof;

Some of the embodiments of the methods and compositions provided herein are methods of producing any of the antigen-specific artificial immunoregulatory T (airT) cells, wherein the antigen-specific artificial immunoregulatory T (airT) cells (airT) cells comprising performing any of the above methods.

In some embodiments, a method of treating a subject with a disease requiring antigen-specific immunosuppression, or suppressing or ameliorating said disease, comprises administering an antigen requiring antigen-specific immunosuppression. administering to said subject a therapeutically effective amount of a plurality of said artificial immunoregulatory T (airT) cells expressing at least one specifically recognizing T cell receptor (TCR). In some embodiments, the disease requiring antigen-specific immunosuppression is an autoimmune disease, an allergic disease or an inflammatory disease.
In some embodiments,
(i) said autoimmune disease is selected from type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, Crohn's disease, bullous pemphigoid, pemphigus vulgaris and autoimmune hepatitis be;
(ii) said allergic disease is selected from allergic asthma, pollen allergy, food allergy, drug hypersensitivity and contact dermatitis;
(iii) said inflammatory disease is selected from islet cell transplantation, asthma, hepatitis, inflammatory bowel disease (IBD), ulcerative colitis, graft versus host disease (GVHD), transplant tolerance induction, transplant rejection and sepsis be done.
In some embodiments,
(i) the antigen associated with the pathogenesis of said autoimmune disease is selected from the autoantigens described in any of Figures 141-144;
(ii) the antigen associated with the etiology of the allergic disease is selected from allergic antigens according to any one of Figures 141-144;
(iii) the antigen associated with the pathogenesis of said inflammatory disease is selected from the inflammation-associated antigens described in any of Figures 141-144;

Some of the embodiments of the methods and compositions provided herein are methods of treating or alleviating a subject with a disorder, comprising administering to said subject any of said artificial immunoregulatory T (airT) cells including a method comprising:

In some embodiments, the disorder is type 1 diabetes, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, rheumatoid arthritis (RA), Crohn's disease, bullous pemphigoid, vulgaris vulgaris. Allergic diseases such as smallpox, autoimmune hepatitis; allergic diseases selected from allergic asthma, pollen allergy, food allergy, drug hypersensitivity and contact dermatitis; and pancreatic islet cell transplantation, asthma, hepatitis, inflammatory bowel disease (IBD) ), an inflammatory disease selected from ulcerative colitis, graft-versus-host disease (GVHD), transplant tolerance induction, transplant rejection and sepsis. In some embodiments, the TCR polypeptide binds an antigen associated with a disorder selected from type 1 diabetes, multiple sclerosis, myocarditis, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE).

In some embodiments, the TCR polypeptide is from the group consisting of vimentin, aggrecan, cartilage intermediate layer protein (CILP), preproinsulin, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) and enolase. Binds an antigen of choice. In some embodiments, the TCR polypeptide binds an antigen comprising an epitope having an amino acid sequence set forth in any of SEQ ID NOS:1363-1376 and 1408-1415.

In some embodiments, the TCR polypeptide has a CD3α polypeptide having an amino acid sequence set forth in any of SEQ ID NOs:1377-1390; and/or an amino acid sequence set forth in any of SEQ ID NOs:1377-1390 It contains a CD3β polypeptide.

It relates to generating airT cells from human CD4+ T cells using gene editing.

Figures 1A, 1B and 1C show exemplary schemas for converting CD4+ T cells into airT cells according to the present disclosure. FIG. 1A is a schematic representation of the FOXP3 locus before (top) and after (bottom) gene editing using FOXP3 TALENs or using FOXP3 guide RNA and CRISPR/Cas9. TALENs or CRISPR/Cas9 cut exon 1 of the FoxP3 locus to initiate site-specific double-stranded DNA cleavage. AAV provides a donor template containing MND and GFP (to analyze editing efficiency), which is inserted into the exon 1 DNA cleavage site. After homologous recombination repair, the MND promoter induces FoxP3 and GFP reporter expression. FIG. 1B shows the timeline of the gene editing and cell analysis steps and the efficiency in generating airT cells from input normal T cells (Tconv). FIG. 1C shows a typical flow cytometry plot showing the correlation between Foxp3 and GFP at 4 days after gene editing. The three plots on the right side of the figure show the expression of CD25, CD127, Helios, CD45RO, ICOS and CTLA-4 in Foxp3+GFP+ gated cells, respectively.

Shown are flow cytometry plots (bottom) showing expression of GFP and Foxp3 at days 4 and 11 after gene editing according to the timeline shown at the top. These data indicated that Foxp3 was efficiently edited in CD4+ T cells, resulting in stable high expression of Foxp3.

Figures 3A, 3B, 3C and 3D show data comparing airT cells with activated naive regulatory T (nTreg) cells. FIG. 3A shows a timeline of steps to generate edTreg cells and activated nTreg cells for comparison. CD4+ cells were isolated from PBMC using MACS CD4+ isolation kit and Tconv cells (CD25-CD127+) and Treg cells (CD25 ^high CD127-) were sorted by flow cytometry. Sorted Tconv and Treg cells were activated with CD3/CD28-activated beads for 48 hours before removing the beads. EdTreg/airT cells were obtained by editing only Foxp3 in Tconv cells using Cas9/Foxp3 gRNA and AAV-MND-LNGFR-Foxp3 ki. nTreg cells were treated similarly except that Foxp3 was not edited. On day 10, LNGFR+ cells were enriched from Foxp3-edited Tconv cells using MACS LNGFR beads. Suppression assays were performed using LNGFR+edTreg and nTreg cells. FIG. 3B shows a comparison of efficacy in generating edTreg or nTreg cells from 1×10 ⁷ PBMC. On day 0, 1×10 ⁷ PBMC were obtained. When Tconv cells and nTreg cells were activated on day 0 and expanded, Tconv cells increased 10- to 30-fold and nTreg cells increased 1- to 2-fold from day 0 to day 10. The yield of Treg cells on day 10 of generation of edTreg cells was calculated based on the editing rate (10-30%). FIG. 3C shows representative flow cytometry plots showing the Treg phenotype in Foxp3-edited Tconv or nTreg cells at day 10. FIG. Top, leftmost panel shows expression of LNGFR in edited Tconv cells, right panel shows Foxp3, Helios, CD25 in edited Treg cells (gated on LNGFR+, top panel) or nTreg cells (bottom panel). , shows the expression of CD127, ICOS and CTLA-4. The upper panel of FIG. 3D shows a comparison of expression levels of Foxp3, CTLA-4 and ICOS in edTreg/airT cells (blue) and nTreg cells (red). The lower table in Figure 3D shows the MFI.

Figures 4A and 4B show that airT cells have superior suppressive activity in vitro than nTreg cells. FIG. 4A shows data from an in vitro suppression assay comparing the suppressive activity of edTreg/air T cells and nTreg cells against CD4+ T _eff cells at the Treg to T _eff ratios indicated in each graph. airT cells and nTreg cells were labeled with EF670, and CD4+T _eff cells were labeled with Cell Trace Violet (CTV). _{airT cells} or nTreg cells and T _eff cells were co-cultured. CD3/CD28-activated beads were added at a ratio of 1:25 (beads:T _eff cells=) and incubated for 4 days, and the cells were analyzed by flow cytometry. Dilution of CTV in T _eff cells was measured as an index of proliferation. FIG. 4B shows the suppression rate calculated by the following formula: (T _eff cells alone + proliferation rate on beads (%)−proliferation rate of T _eff cells co-cultured with Treg cells (%))/(T _eff Growth rate (%) on cells only + beads) x 100.

A representative islet-specific TCR lentiviral construct expressing a rare islet-specific TCR from a subject with type 1 diabetes (T1D) is shown. Panel A shows lentiviral vectors encoding TCRs (4.13, T1D2, T1D4, T1D5-1 or T1D5-2) specific for GAD65 or IGRP, their epitope specificities, and the TCR α or β chains used. shows a table. Panel B shows the structure of an islet-specific TCR expressed by lentivirus. These TCR constructs contain a human TCR variable region derived from an islet-specific TCR and a mouse TCR constant region, resulting in improved pairing of human TCR chains when transduced.

In order to verify the expression of pancreatic islet antigen-specific TCR, the results of analyzing the expression of mouse TCRβ and proliferation of pancreatic islet antigen-specific T cells are shown. Figure A shows flow cytometry plots of CD4+ T cells activated with CD3/CD28 beads after isolation and transduced with lentivirus encoding islet antigen-specific TCRs. These flow cytometry plots analyzed mTCRβ expression by gating on CD3/CD28-activated CD4+ cells 9 days after transduction with lentivirus (LV) encoding an islet-specific TCR. indicates Panel B shows CD4+ T cells transduced with a lentivirus encoding an islet-specific TCR were labeled with Cell Trace Violet (CTV) and APCs (radioactive The results of co-culture with irradiated PBMC) for 5 days and analysis by flow cytometry are shown. These flow cytometry plots show that lentivirally transduced CTV-labeled CD4+ T cells were compared with antigen-presenting cells (APCs; irradiated PBMCs) in the presence of peptides recognized by the TCR or irrelevant peptides. The results of co-culturing for days and analyzing the cell proliferation by flow cytometry are shown. Dilution of CTV was taken as an index of proliferation.

Generation of Foxp3-edited T cells with islet-specific TCR. FIG. A shows the timeline of steps to generate edTreg cells with islet-specific TCRs. Panel B shows typical flow cytometry plots showing expression of mTCRβ and LNGFR/Foxp3 in CD4+ cells 7 days after T1D4 TCR or T1D5-1 TCR transduction and Foxp3 editing. . The right panel shows the analysis of expression of CD25, CD127, CTLA-4, and ICOS by gating on LNGFR+ cells.

Concerning exemplary antigen-specific suppression assays according to the present disclosure. FIG. A shows the timeline for generating edTreg cells expressing islet-specific TCRs. Using MACS LNGFR beads, edTreg cells expressing islet-specific TCRs (non-lentiviral-derived TCRs; T1D4 TCR or T1D5-1 TCR) were enriched based on LNGFR expression. The resulting LNGFR+ cells were aliquoted and cryopreserved until the next experiment. FIG. B shows a summary of the evaluation method for the antigen-specific suppression assay. CD4+ T cells transduced with islet-specific TCR (T1D4 TCR or T1D5-1 TCR) were used as T _eff cells. T _eff and T reg cells were labeled with separate reagents (e.g. CTV or EF670) and ed T reg cells were used to label T _eff and T reg cells in the presence of APCs (irradiated autologous PBMC) and various peptides. T _eff cells were cultured alone without edTreg cells, either co-cultured at a 1:1 ratio or a 1:2 ratio. After 1 or 4 days of incubation, cells were stained and analyzed by flow cytometry to measure cytokine production and T _eff cell proliferation.

FIG. 10 shows the suppressive activity of edTreg/airT cells against the proliferation of T _eff cells in the presence of the peptides indicated in the graph and APC. T _eff cells were labeled with CTV and Treg cells with EF670. CD4 T cells transduced with T1D4-TCR (T1D4 T _eff ) were treated with various peptides (DMSO, IGRP 241, IGRP 305 or IGRP241 + IGRP 305) and edTreg cells expressing T1D4-TCR (T1D4 edTreg) or T1D5-1-TCR-expressing edTreg cells (T1D5-1 edTreg), or T1D4 T _eff cells were cultured alone without edTreg cells. After 4 days of co-cultivation, cells were stained and T _eff cell proliferation was analyzed from CTV dilutions. Flow cytometry plots show proliferation of T _eff cells gated on CD3+CD4+CTV+EF670-LNGFR-.

edTreg/airT cells suppress cytokine production in T _eff cells. T _eff cells were labeled with CTV and Treg cells with EF670. Co-culture of untransduced edTreg cells or T1D4-transduced edTreg/airT cells with T1D4 T _eff cells in the presence of APC and peptide (DMSO or IGRP 241) or without edTreg cells T1D4 T _eff cells were cultured alone. After 1 day of co-culture, cells were contacted with BFA for 4 hours, stained and analyzed for cytokine production from T _eff cells. Flow cytometry plots show TNF, IFNγ or IL-17 production from CD4+CTV+EF670− gated T1D4 T _eff cells.

Fig. 2 shows the generation and characterization of human Foxp3-edited antigen-specific human CD4+ T cells.

A typical scheme for generating antigen-specific human edTreg/air T cells from peripheral blood cells (upper panel) and phenotypes of FOXP3-edited antigen-specific human CD4+ T cells (lower panel) are shown. The lower panel shows representative GFP expression in tetramer-positive (Tmr+; a mixture of influenza antigenic peptide-MHC class II tetramers and tetanus toxoid antigenic peptide-MHC class II tetramers) human CD4+ T cells 4 days after gene editing. Flow cytometry plots (left) and percentages (right) are shown (n=5).

Characterization of FOXP3-edited antigen-specific human CD4+ T cells. Panel A shows the phenotype of FOXP3-edited antigen-specific human CD4+ T cells. Flow cytometry data were summarized in bar graphs (n=5). This bar graph shows the expression of each Treg marker and intracellular IL-2 production in Tmr+edTreg cells, Tmr+mock-edited cells, and thymus-derived Treg cells (tTreg) obtained from unrelated donors. Data shown in the graph are representative of 5 independent experiments. P-values indicating statistically significant differences are indicated above each bar. Panel B shows that antigen-specific human edTreg/air T cells suppress proliferation of T _eff cells in vitro. Suppression assays were performed by co-culturing Tmr+edTreg/air T cells or mock-edited Tmr+ cells with T _eff cells from healthy controls, antigen presenting cells (APCs), and soluble anti-CD3 and anti-CD28 antibodies. The ratio of APC (irradiated CD4-PBMC) to Tmr+ _edTreg cells or mock-edited Tmr+ cells to Teff cells was 2:1:1. 1 μCi of ³ H was added 18 hours before the end of the 4-day assay and cell proliferation was measured in a scintillation counter. Each bar graph represents the mean of results from three experiments with three donors.

We show successful generation of antigen-specific edTreg/air T cells by editing Foxp3 after stimulation with peptide. FIG. A shows the timeline of the antigen-specific T cell expansion and gene editing steps. To expand T cells specific for MP peptide, HA peptide, or tetanus toxoid peptide (TT), cells were stimulated with peptide pools for 9 days, then activated with CD3/CD28-activated beads for gene editing. . To enhance the expansion of antigen-specific Treg cells, beads were added to flow cytometrically sorted cells. Panel B shows flow cytometry plots showing GFP and Foxp3 expression 15 days after gene editing. GFP+Foxp3+ cells were CD25+CD127-, and tetramer staining showed that approximately 60% of these were specific for MP, HA or TT peptides.

Antigen-specific suppression by Foxp3-edited Treg/air T cells. Figure A shows the timeline of the steps to generate antigen-specific edTreg/air T cells for use in suppression assays. Fifteen days after gene editing, GFP+ cells were sorted by flow cytometry, activated with CD3/CD28 beads and expanded. After 7 days of incubation, the beads were removed and the edTreg/airT cells were collected. Suppression assays were performed using edTreg/airT cells expanded for 11 days. FIG. B shows an overview of the inhibition assay design. CD4+CD25+ cells were isolated from autologous PBMC, labeled with EF670 and used as T _eff cells. Also, irradiated CD4-CD25+ cells were used as APCs. edTreg/airT cells were labeled with Cell Trace Violet (CTV). T _eff cells, APCs and edTreg/airT cells were co-cultured in the presence of DMSO or peptide pools (MP+HA+TT), or T _eff cells and APCs were co-cultured without edTreg/airT cells. Panel C shows the results of staining cells and analyzing by flow cytometry after 7 days of co-culture. CD3+CD4+EF670+CTV- cells were gated as T _eff cells. Panel D shows proliferation of T _eff cells measured from the dilution of EF670 in T _eff cells and normalized to EF670- cells which accounted for 15% in co-cultures of T _eff cells, APCs and peptide pools as 100% proliferation rate. Shows proliferation rate. Inhibition rate was calculated as 100-proliferation rate (%).

It shows that islet-specific T cells with multiple specificities can be expanded by stimulation with peptides. Figure A shows a typical timeline for generating islet antigen-specific edTreg/air T cells. Freshly isolated CD4+CD25- cells were stimulated with islet-specific peptide pools and APCs (irradiated autologous CD4-CD25+ cells) for 14 days, and on day 13, tetramer staining of islet-specific T cell proliferation was performed. analyzed by Panel B shows flow cytometry plots of islet-specific T cells stained with each tetramer or pool of tetramers and gated on CD4+ cells.

Generation of islet-specific Treg cells with multiple specificities is shown. Panel A shows islet-specific T cells were stained with tetramer and sorted by flow cytometry on day 14. FIG. Sorted tetramer-positive cells were activated with CD3/CD28 beads for 72 hours prior to Foxp3 editing. Cells were stained and analyzed 3 days after editing. Flow cytometry plots show Foxp3 and LNGFR expression in mock or Foxp3-edited cells (left) and CD25, CD127 and CD45RO expression in LNGFR+ gated cells (right). Panel B shows cells stained with each tetramer or pool of tetramers and shows flow cytometry plots showing tetramer-positive cells in Foxp3-edited LNGFR+Foxp3+ cells.

Double editing of human CD4+ T cells targeting two alleles results in inactivation of the endogenous TCR resulting in artificial Treg cells expressing Foxp3 and an antigen-specific TCR.

Schematic representation of a typical CD4+ T cell gene-edited to express an exogenous antigen-specific TCR without expressing an endogenous TCR and to have a Treg phenotype. In this scheme, conversion of conventional CD4+ T cells to antigen-specific Treg cells involves the following three genetic changes. 1) stably expressing the FOXP3 transcription factor in CD4+ T cells to induce the Treg phenotype; stably expressed to induce Treg immunosuppressive activity; and 3) genetic deletion of the endogenous T cell receptor (TCR) so that immunosuppressive function is directed only to the desired antigen. thing.

Typical AAV constructs used for CRISPR-assisted gene editing in human and mouse TRAC loci are shown. This list shows the adeno-associated virus plasmid constructs generated for CRISPR-mediated homologous recombination repair, which were built on the relevant gRNAs. A number is assigned to each of them.

Exemplary CRISPR-based targeting of the human TRAC locus for knockout/knockin is shown. More specifically, a schematic representation of the human TRAC locus showing the relative positions of the four gRNA sequences tested (PC_TRAC_E1_gRNA1 to PC_TRAC_E1_gRNA4) is shown. Exon 1 of the TRAC locus is indicated by the bottommost bar and extends from around position 1160 to above position 1400. Common SNPs are shown near 1160th and 1400th. The position of the previously reported positive control gRNA sequence (TCRa G4old) is shown around position 1320.

Analysis of the suitability of each guide RNA (gRNA) for non-homologous end joining (NHEJ) to knock out CD3 in human CD4+ primary T cells. Data were obtained by FACS analysis. Figure A. Flow cytometry showing the expression of CD3 in mock-edited CD4+ T cells or CD4+ T cells in which the TCR locus was edited using each of the four guide RNAs two days after gene editing. A metric plot is shown. As a control, TCRa_G4old, which has been previously demonstrated to be able to knock out CD3 expression, was used. FIG. B shows a histogram showing the CD3 knockout rate.

The results of examining the frequency of indels by analysis using ICE (Inference of CRISPR Edits) are shown. On-target site-specific activity was measured by ICE (Inference of CRISPR Edits) and confirmed that indels were specifically induced by gRNA_1 and gRNA_4 at the TRAC locus compared to predicted off-target sites did it.

ICE analysis of predicted off-target sites of gRNAs targeting the TRAC locus is shown. The induction frequency of indels at the top three predicted off-target sites of TRAC gRNA 1 and TRAC gRNA 2 (predicted based on the frequency of mismatches and their positions) was examined by sequence deconvolution analysis using ICE. .

A typical experiment with double editing using AAV to assess knock-in to two alleles is outlined. A. A schematic diagram of each AAV construct used in this experiment is shown. GFP/BFP expression is induced by the MND promoter after gene editing. B. A timeline of the experimental procedure is shown. CD4+ T cells were stimulated with CD3/CD28 beads for 3 days prior to gene editing. GFP and BFP expression in cells was assessed by flow cytometry 3 and 6 days after gene editing.

Figure 2 shows that double-editing the TRAC locus in human CD4+ cells yields a double-positive cell population. Panel A, Expression of GFP and BFP in mock-edited cells 2 days after gene editing and in cells edited with a mixture of MND.GFP and MND.BFP (10% No.3207 AAV virus + 10% No.3208 AAV virus). Flow cytometry plots showing . The virus titer of No.3207 was 3.3×10 ¹² and the virus titer of No.3208 was 2.53×10 ¹² . Panel B shows histograms showing the percentage of double-negative, GFP-only positive, mCherry-only-positive and GFP/mCherry double-positive cells in the double-edited cells.

Schematic showing a homologous recombination repair (HDR) knock-in construct containing a typical split IL-2 CISC for selection of double editing cells. In the construct shown, the CISC (chemoinducible signaling complex) is split into two separate constructs, with each CISC component co-expressed with a different reporter (GFP or mCherry in this case). Each construct contains a rapamycin-binding complex half (either an FKBP or FRB domain) linked to a chimeric endoplasmic reticulum targeting domain, wherein the FKBP or FRB domain is a transmembrane domain and a cell domain. It is fused to one half of the IL-2R signaling complex (IL-2RB or IL-2RG), which consists of the endodomain. By delivering the cDNA encoding each CISC component co-expressed with a GFP- or mCherry-tag into primary human CD4+ T cells, we generated dual-edited cells containing both CISC components and thus also GFP and BFP. It can be selectively propagated.

Shown is a typical timeline of double editing of CD4+ T cells using AAV, expansion in the presence of rapalogs, and analysis of enriched cells. Cells were stimulated with CD3/CD28 beads for 3 days prior to gene editing. Two days after gene editing, cells were analyzed for GFP and mCherry expression by flow cytometry and cells were expanded in medium containing 50 ng/ml human IL-2 or 100 nM rapalog. Enrichment of GFP+mCherry+ double-positive cells was assessed by flow cytometry 6, 8 and 10 days after gene editing.

A FACS analysis examining the initial double-editing rate is shown. Panel A, mock-edited cells, cells edited with MND.GFP.FRB.IL-2RB (No.3207 AAV 20%), edited with MND.mCherry.FKBP.IL-2RB (No.3208 AAV 20%) Flow showing GFP and mCherry expression in cells and cells edited with a mixture of MND.GFP.FRB.IL-2RB and MND.mCherry.FKBP.IL-2RB (No.3207 10% + No.3208 10%). Cytometry plots are shown. The virus titer of No.3207 was 3.3×10 ¹² and the virus titer of No.3208 was 2.53×10 ¹² . Panel B shows histograms showing the percentage of double-negative, GFP-positive, mCherry-positive and GFP/mCherry double-positive cells in double-edited cells.

Representative data showing enrichment of double-edited cells with rapalogs are shown. Panel A, Expression of GFP and BFP in mock-edited cells 2 days after gene editing and in cells edited with a mixture of MND.GFP and MND.BFP (10% No.3207 AAV virus + 10% No.3208 AAV virus). Flow cytometry plots showing . The virus titer of No.3207 was 3.3×10 ¹² and the virus titer of No.3208 was 2.53×10 ¹² . Panel B shows histograms showing the percentage of double-negative, GFP-only positive, mCherry-only-positive and GFP/mCherry double-positive cells in double-edited cells.

Histograms showing the percentage of double-negative, GFP-only positive, and mCherry-only positive cells after exposure to IL-2 or rapalog are shown. These data indicated that treatment with rapalogs did not significantly alter the proportions of single positive and unedited cell populations.

Data from the first double-edited FACS analysis using two different donors are shown. Figure A shows the timeline of the gene editing and analysis steps. Panel B shows histograms showing the percentage of double-negative, GFP-positive, mCherry-positive and GFP/mCherry double-positive cells in double-edited cells from each donor. Donor R003657 was a 28 year old Caucasian male and donor R003471 was a 29 year old Caucasian male.

Data from FACS analysis in which two alleles of cells from donor R003471 were gene edited and enriched in the presence of rapalogs are shown. Figure A shows flow cytometry plots showing GFP and mCherry expression after enrichment for 5 days in the presence of rapalogs. Panel B shows histograms showing the percentage of GFP/mCherry double positive cells after growth in the presence of IL-2 or rapalogs.

Schematic showing a typical split-type CISC construct for inserting TCR and Foxp3 and enriching for double-edited cells. CISC is divided into two different constructs and each CISC component is co-expressed with an antigen-specific TCR (typical T1D4 TCR shown in schematic) or Foxp3. Each construct contains a rapamycin-binding complex half (either an FKBP or FRB domain) linked to a chimeric endoplasmic reticulum targeting domain, wherein the FKBP or FRB domain is a transmembrane domain and a cell domain. It is fused to one half of the IL-2R signaling complex (IL-2RB or IL-2RG), which consists of the endodomain. By delivering cDNAs encoding each CISC component co-expressed with the T1D4 TCR/Foxp3 into primary human CD4+ T cells, dual-edited cells containing both CISC components and thus also T1D4 TCR and Foxp3 were generated. It can be selectively propagated.

It relates to the preparation of reagents for evaluating the function of antigen-specific airT cells in in vivo models of autoimmune diseases.

Schematic representation of the mouse TRAC locus showing the relative positions of the three novel gRNA sequences tested (PC_mmTrac_E1_gRNA1 to PC_mmTrac_E1_gRNA3). Exon 1 of the TRAC locus is shown in blue.

Data from FACS analysis examining CD3 knockout in mouse CD4+ T cells are shown. (A) Flow cytometry showing the expression of mouse CD3 in mock-edited CD4+ T cells or CD4+ T cells in which the TCR locus was edited using each of the three guides, 2 days after gene editing. A metric plot is shown. FIG. B shows a histogram showing the mCD3 knockout rate when using each guide RNA.

A typical experiment with double editing using AAV to assess knock-in to two alleles is outlined. Panel A shows a schematic of each AAV construct used in this experiment. GFP/BFP expression is induced by the MND promoter after gene editing. B. A timeline of the experimental procedure is shown. Mouse CD4+ T cells were stimulated with CD3/CD28 beads for 3 days prior to gene editing. GFP and BFP expression in cells was evaluated by flow cytometry 3 and 5 days after gene editing.

Data from FACS analysis examining single and double editing rates at the mouse TCRα locus are shown. Flow cytometry plots show mock-edited cells, cells edited with MND.GFP (No.3211 10%), cells edited with MND.BFP (No.3212 10%), and a mixture of MND.GFP and MND.BFP. GFP and BFP expression 3 days after gene editing in cells edited with (No.3207 5% + No.3208 5%). Cells edited with the mixture had a total percentage of GFP/BFP double positive cells of 1.97%.

It relates to the function of airT cells in antigen-specific in vivo conditions.

To test whether MOG-specific edTreg/air T cells (indicated in white) can suppress effector T cells (T _eff ) in an experimental autoimmune encephalomyelitis mouse model, a multiple sclerosis model. shows a schematic diagram of the experimental design for

It relates to experiments showing that mouse FOXP3 TALENs catalyze efficient disruption of FOXP3 to initiate nondisruptive donor template recombination. Panel A shows the binding sites of the FOXP3 TALEN pair in the human FOXP3 gene. Panel B shows the target binding sites of mouse FOXP3 TALEN pairs in the mouse FOXP3 gene. Panel C. Human CD4+ T cells (left) and mouse CD4 cells 5-7 days after transfection with control mRNA (encoding blue fluorescent protein) or mRNA encoding TALENs specific for human FOXP3 or mouse FoxP3. Shown is the frequency of indels at FOXP3 TALEN cleavage sites in + T cells (right). Each graph shows the average frequency of indels analyzed by colony sequencing of amplicons amplified by PCR around the gDNA target site. Sequences were determined from 20-40 colonies for each experiment.

Generation of edTreg/air T cells from antigen-specific mouse CD4+ T cells. Panel A shows a schematic representation of the FOXP3 locus after gene editing using mouse FOXP3 TALENs and AAV donor templates for mouse FOXP3 MND-GFP knock-in (ki). Upon gene editing, the MND promoter drives expression of the chimeric GFP-FoxP3 protein. Panel B shows flow cytometry plots showing expression of GFP in antigen-specific mouse CD4+ T cells 2 days after gene editing. Panel C shows the average percentage of GFP+ cells in multiple experiments (n=10). Panel D shows flow cytometry plots showing expression of Treg markers associated with mouse edTreg/air T cells.

Fig. 2 shows evaluation results comparing the functions of antigen-specific edTreg/airT cells and polyclonal edTreg/airT cells in a mouse model of multiple sclerosis. Figure A shows flow cytometry plots showing expression of GFP in MOG-specific and polyclonal mouse CD4+ T cells after FACS sorting 2 days after gene editing. FIG. B shows a schematic showing the in vivo experimental design and timeline using the experimental autoimmune encephalomyelitis (EAE) mouse model. 2D2 (MOG-specific) T _eff cells (30,000) were delivered to RAG1−/− recipient mice simultaneously with edTreg/air T cells (30,000) generated from 2D2 or C57Bl/6 mice. Alternatively, 2D2 (MOG-specific) T _eff cells (30,000) alone were delivered to RAG1−/− recipient mice without transfer of edTreg/air T cells. Both mouse strains were on the C57Bl/6 genetic background. Analysis was performed on day 7.

Data showing that antigen-specific edTreg/air T cells delay T _eff cell proliferation, activation and cytokine production. The immunophenotype of T cells obtained from inguinal and axillary lymph nodes of recipient mice 7 days after cell transfer was evaluated by flow cytometry. CD45+ indicates the results detected with panCD45 antibody (antibody that recognizes all CD45 isoforms and both CD45.1 alloantigen and CD45.2 alloantigen). (A) total number of CD45+CD4+ cells, (B) total number of other T cell subsets indicated in the graph, and (C) proliferation of GFP+ cells. Each data represents the results of three independent experiments. Each bar graph represents the mean±SD. P-values indicating statistically significant differences are indicated above each bar graph.

Figure 2 shows data demonstrating that antigen-specific edTreg/air T cells suppress proliferation of T _eff cells in vivo. Panel A. Actively dividing cells were labeled by administering the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU) to the selected mice 2 hours prior to sacrifice. Flow cytometry plots are shown. EdU uptake by T cells was measured by intracellular labeling with anti-EdU antibody and flow cytometry. Each flow cytometry plot was obtained by analyzing T cells isolated from lymph nodes 7 days after cell transfer. Panel B shows a bar graph summarizing the mean percentage of cells that have taken up EdU in various cell subsets. Panel C shows a bar graph summarizing the percentage of GFP+ lymphocytes. Each flow cytometry plot shows representative results of at least three independent experiments. Each bar graph represents the mean±SD. P-values indicating statistically significant differences are indicated above each bar graph.

It relates to experiments investigating the function of antigen-specific T cells in an adoptive transfer NSG mouse model with type 1 diabetes. Antigen-specific (BDC) recombinant edTreg/airT cells or polyclonal (NOD) recombinant edTreg/airT cells, or antigen-specific nTreg cells were injected into NSG mice followed by antigen-specific T _eff cells. Mice were monitored for diabetes up to 90 days after injection. The graph shows the percentage of mice that developed diabetes after administration of the indicated mock-edited, Foxp3-edited or nTreg cells and effector cells from NOD or BDC2.5 mice.

Editing of Foxp3 in CD4+ T cells from antigen-specific NOD mice. Figure A shows the efficiency of cleavage by CAS9/CRISPR RNPs using various guide RNAs in BDC2.5 NOD mice. Panel B shows the repair template delivered by AAV5. Upon gene editing, the MND promoter drives expression of the chimeric GFP-FoxP3 protein. (C) Flow cytometry plots showing expression of GFP in mock-edited antigen-specific mouse CD4+ T cells and antigen-specific mouse CD4+ T cells edited with the GFP-Foxp3 construct at 2 days post-editing. .

Phenotype of FOXP3-edited antigen-specific NOD CD4+ T cells. Left panel: Flow cytometry plots showing GFP and Foxp3 expression in edited cells. Middle panel: IL-2, IFN- in antigen-specific mouse NOD CD4+ T cells edited with GFP-Foxp3 constructs (top plot) and mock-edited antigen-specific mouse NOD CD4+ T cells (bottom plot). Flow cytometry plots showing expression of γ and IL-4 are shown. Right panel: histogram showing percentage of cells positive for IL-2, IFN-γ and IL-4 4 days after editing.

It relates to an experiment examining the phenotype of cells transferred into the adoptive transfer NSG mouse model. Figure A shows the experimental design showing the number and type of cells administered to each group of mice. Panel B shows flow cytometry plots showing the phenotypes of T _eff , edTreg/airT and nTreg cells injected into NSG mice.

Antigen-specific T cell function in an adoptive transfer NSG mouse model. Figure A shows the experimental design. Antigen-specific (BDC) recombinant edTreg/airT cells or polyclonal (NOD) recombinant edTreg/airT cells, or antigen-specific nTreg cells were injected into NSG mice followed by antigen-specific T _eff cells. Mice were monitored for diabetes up to 90 days after injection. FIG. B shows a graph showing the percentage of mice that developed diabetes after administration of the indicated mock-edited, Foxp3-edited or nTreg cells and effector cells from NOD or BDC2.5 mice. Antigen-specific edTreg/airT cells were significantly more protective against type 1 diabetes than mock-edited T cells, polyclonal edTreg/airT cells, or polyclonal nTreg cells.

Concerning the construction of a mouse AAV donor template design for making airT cell products containing a selectable marker (LNGFR).

A typical repair template used for editing mouse Foxp3 is shown. Stable expression of Foxp3 by an AAV promoter-LNGF.P2A knock-in construct was tested in mouse T cells.

Phenotypes of mouse edTreg/air T cells using different homologous donor cassettes. Flow cytometry plots show expression of LNGFR, FOXP3, CD25 and CTLA-4 in mock-edited cells or cells edited with MND.LNGFR.P2A KI (No.3189) or PGK.LNGFR.P2A KI (No.3227). indicates

Data showing editing rate and LNGFR expression in gene-edited mouse Treg/airT cells are shown. Flow cytometry plots show expression of LNGFR and GFP in mock-edited cells or cells edited with MND-GFPki (No.1331) or MND.LNGFR.P2A.KI (No.3189).

Data are shown using an anti-LNGFR column to enrich for gene-edited LNGFR+ T cells from B6 mice. Flow cytometry plots show the expression of LNGFR in cells prior to purification on the Miltenyi anti-LNGFR column, cells in the flow-through, and cells eluted from the column.

A comparison of human CD4 T cells edited with FOXP3 and human CD4 T cells transduced with FOXP3 lentivirus (LV) is shown. FIG. A shows a schematic representation of the LV construct. The MND promoter drives expression of a transcript encoding the same GFP-FOXP3 fusion protein contained in airT cells. This transcript contains WPRE and poly(A) signals for efficient nuclear transport and mRNA stability. Below is a representative flow showing FOXP3 and GFP expression in mock-edited T cells, or FACS sorted tTreg cells (CD4+CD25++CD127-), airT cells or LV Treg cells (CD4+GFP+). Cytometry plot. All of these cells were expanded in vitro for more than 14 days while being stimulated with CD3/CD28 beads. Panel B shows the mean viral copy number (±SD) of FACS-sorted cells after transduction with LV (left side; n=6). Scatter plots (right) show the MFI of the GFP+ population in each sample (n=5; P-values determined by two-tailed Student's t-test are shown). FIG. C shows bar graphs showing the mean percentage of each cell analyzed by flow cytometry staining for each protein indicated (upper graph) and MFI (lower graph). Live singlet cells were further gated on CD4+GFP+ (LV Treg and edTreg), CD4+FOXP3+ (tTreg) or CD4+ (mock). For markers with a clear bimodal distribution, only the MFI of the positive population was calculated. Error bars indicate ±SD. Routine two-way ANOVA was performed and P-values were corrected with Tukey's multiple comparison test. P-values shown in black show comparisons with mock-edited cells, and P-values shown in red show comparisons with groups shown with dashed lines. Panel D shows the percent suppression as a function of dilution of Treg cells or mock cells (upper panel). In addition, histograms of growth pigments when T reg cells or mock cells and T _eff cells were cultured at various ratios are shown (lower figure). Suppression rate (%) = [(percentage of dividing cells in the absence of Treg cells (%) - percentage of dividing cells in the presence of Treg cells (%)) / percentage of dividing cells in the absence of Treg cells ( %)] x 100. FIG. E shows plots showing the percentage of GFP+ cells in culture over time data points and their simple linear regression after FACS purification. airT cells (n=4) and LV Treg cells (n=6); data from 6 experiments are shown. Dashed lines indicate 95% confidence intervals. P-values were determined using the F-test.

FIG. 4 shows another schematic and data for an exemplary double-editing method according to the present disclosure for generating drug-selectable, antigen-specific airT cells in which the endogenous TCR has been knocked out.

Schematic showing a double-editing strategy designed to a) eliminate endogenous TCR expression and b) yield selectable antigen-specific airT cells. IL-2 CISC/DISC was split into two (FKBP-IL2RG and FRB-IL2RB), expression cassettes combining one of them with FOXP3, and expression combining the other with an islet antigen-specific TCR candidate (T1D4) The cassette and can be delivered to the same locus (Method 1) or can be delivered to two separate loci (Method 2). Endogenous TCR can be depleted by targeting the TRAC locus of CD4+ T cells. Although Method 2 may result in a higher initial double-editing rate, it requires two nuclease target sites, resulting in two double-strand breaks in the host cell's genome, each of which undergoes homologous recombination repair. done. Method 1 utilizes one nuclease target site to generate one double-strand break.

Schematic representation of AAV HDR donor constructs used for double editing of human T cells. The first seven constructs are split IL-2 CISC repair templates with GFP-, mCherry-, HA-tagged FOXP3 or T1D4 driven by the MND promoter. Each component of the split CISC comprises one half of the rapamycin-binding and heterodimerizing complex (one of the FKBP or FRB domains) and linked to it a chimeric endoplasmic reticulum targeting domain, The FKBP or FRB domain is fused to one half of the IL-2R signaling complex (IL-2RB or IL-2RG), which consists of transmembrane and intracellular domains. Each repair template is flanked by 300 bp homologous arms that match the gRNA (gRNA_4) targeting the TRAC locus or the gRNA (gRNA_T9) targeting the FOXP3 locus (No.3207, No.3208, No. 3240, No. 3243, No. 3251, No. 3252, No. 3273). The following four constructs (No. 3253, No. 3258, No. 3292, No. 0001) were used to knock-in in-frame the TCR cassette containing the CISC components but no promoter, and the TRAC locus. (gRNA_1). The last two constructs (No. 3280 and No. 3262) encode free FRB domains that sequester rapamycin in the cytoplasm, thereby eliminating or reducing the negative effects of rapamycin on gene-edited cells. Split DISC repair template containing cDNA and CISC components. These two constructs additionally contain mCherry or FOXP3 driven by the MND promoter.

Figure 3 shows the double-editing rate of the human TRAC locus in CISC construct-edited CD4+ T cells from donor R003657 with or without rapalog selection. Figure A shows the timeline of gene editing, enrichment and analysis steps of CD4+ T cells from donor R003657 using AAV No.3207 and AAV No.3208 (by co-delivery of RNP and AAV). (B) Flow cytometry plots comparing the percentage of initial GFP/mCherry double-positive cells in mock-edited and double-edited samples and enriched for 7 days in the presence of IL-2 or rapalog (AP21967). Flow cytometry plots showing the percentage of GFP/mCherry double-positive cells are shown. Panel C compares the proportions of double-negative, GFP-positive, mCherry-positive and GFP/mCherry double-positive cells in double-edited cells after IL-2 enrichment versus rapalog enrichment. Histograms are shown.

FIG. 4 shows the double editing rate of the TRAC locus when CD4+ T cells from donor R003471 edited with CISC constructs are selected with rapalogs. Figure A shows the timeline of gene editing, enrichment and analysis steps of CD4+ T cells from donor R003471 using AAV No.3207 and AAV No.3208. (B) Flow cytometry plots comparing the percentage of initial GFP/mCherry double-positive cells in mock-edited and double-edited samples and enriched for 7 days in the presence of IL-2 or rapalog (AP21967). Flow cytometry plots showing the percentage of GFP/mCherry double-positive cells are shown. Panel C compares the proportions of double-negative, GFP-positive, mCherry-positive and GFP/mCherry double-positive cells in double-edited cells after IL-2 enrichment versus rapalog enrichment. Histograms are shown.

We show that double editing of the TRAC locus in human CD4+ T cells can generate rapalog-selectable, antigen-specific air T cells. Figure A shows a schematic showing the AAV HDR donor construct used to introduce the 'split' IL-2 CISC component for selection of dual edited cells. The CISC components (IL2RG and IL2RB) are separately incorporated into two constructs and co-expressed with HA-FoxP3 cDNA (AAV No.3240) or islet-specific TCR T1D4 (AAV No.3243). Each repair template is flanked by identical homologous arms that are not cleaved by gRNAs targeting the TRAC locus. Only CD4+ T cells edited by integrating one copy of each construct are expected to be selectively expandable by treatment with rapalogs. Panel B shows the timeline of the double editing steps of CD4+ T cells with AAV/RNP, the expansion steps in the presence of rapalogs, and the analysis steps of the enriched cells, which are important in this study. Cells were stimulated with CD3/CD28 beads for 3 days prior to gene editing. Two days after gene editing, HA-FoxP3 and TCR expression in cells was analyzed by flow cytometry and cells were expanded in media containing 50 ng/ml human IL-2 or 100 nM rapalog. Enrichment of double-positive cells positive for HA-FoxP3 and TCR was assessed by flow cytometry 5 and 8 days after gene editing. Panel C shows enrichment of double-edited cells with rapalogs. Left: Flow cytometry plots analyzing HA-FoxP3 and TCR expression in double-edited cells expanded for 8 days in the presence of IL-2 or rapalogs. Right panel: quantification of the percentage of HA-FoxP3/TCR double-positive cells expanded for 5 or 8 days in the presence of IL-2 or rapalogs.

Shown are data demonstrating that lowering serum concentration improves total and double editing rates of the TRAC locus. FIG. A shows a timeline showing the double editing process of CD4+ T cells with AAV and the expansion process with rapalogs. Gene editing of human CD4+ T cells was performed using TRAC gRNA_4 and AAV No.3243 and AAV No.3240 constructs (double editing at one locus). Immediately after electroporation for RNP delivery, cells were seeded in medium containing 20%, 2.5%, 1% or 0% FBS (recovery medium) and infected with AAV. Approximately 16 hours later, the medium was changed to medium containing 20% FBS and FACS analysis was performed on day 3 to determine the editing rate. Cells recovered in medium containing 2.5% FBS were expanded in the presence of IL-2 or rapalog for an additional 7 days. Panel B shows the expression of T1D4 and FOXP3 in mock-edited cells, single-edited cells and AAV mixture-edited cells (AAV No.3243 10% and AAV No.3240 10%) after 3 days of gene editing. Cytometry plots are shown. AAV No.3243 (pAAV.MND.T1D4.FRB.IL2RB) had a viral titer of 4.2×10 ¹¹ and AAV No.3240 (pAAV.MND.FOXP3-HA.FKBP.IL2RG) had a viral titer of 1.3. × 10 ¹² . Panel C shows histograms showing the percentage of double negative, FOXP3-HA positive, T1D4 positive and FOXP3/T1D4 double positive cells in the double edited cells.

A comparison of IL-2 enrichment versus rapalog enrichment of double edited cell populations is shown. Double editing of the TRAC locus was performed by the method shown in FIG. Panel A shows the expression of T1D4 and FOXP3 when treated with 50 ng/mL IL-2 or 100 nM rapalog (AP21967) for 7 days. Flow cytometry plots compared with edited cells are shown. Data show only conditions using recovery medium containing 2.5% FBS. Panel B shows histograms showing the percentage of double negative, FOXP3-HA positive, T1D4 positive and FOXP3/T1D4 double positive cells in double edited cells after enrichment.

It relates to a method for testing the double-editing method of editing two loci of human CD4+ T cells. Panel A shows a schematic representation of an AAV HDR donor construct designed to introduce a split IL-2 construct used for selection of double-edited cells using a double-editing approach that edits two loci. show. The CISC components are split between the two constructs and co-expressed with mCherry (No.3207) or GFP (No.3251). One repair template is flanked by homologous arms compatible with gRNAs targeting the TRAC locus, and the other repair template is flanked by homologous arms compatible with gRNAs targeting the FOXP3 locus. . Only CD4+ T cells that have been edited with both expression cassettes integrated (at the appropriate loci) are expected to be able to selectively expand under treatment with rapalogs. FIG. B shows a timeline showing the double-editing process of CD4+ T cells with AAV and the expansion process with rapalogs. Using human TRAC gRNA_4 and human FOXP3 gRNA_T9 with AAV construct No.3251 (MND.mCherry.FKBP.IL2RG) and AAV construct No.3207 (MND.GFP.FRB.IL2RB) (editing two loci double editing method), gene editing of human CD4+ T cells was performed. Immediately after electroporation, cells were seeded in medium containing 20% or 2.5% FBS (recovery medium). Approximately 16 hours later, the medium was changed to medium containing 20% FBS and FACS analysis was performed on day 3 to determine the editing rate. Cells recovered in 2.5% FBS-containing media were cultured in the presence of IL-2 or rapalogs for an additional 7 days to monitor enrichment.

We show that recovery in medium containing 2.5% FBS improves double-editing rates measured 3 days after gene editing. A double-editing method for editing two loci was performed by the method shown in FIG. Panel A, GFP and mCherry expression in mock-edited and double-edited cells 3 days after gene editing was restored in recovery medium containing 20% FBS and recovery medium containing 2.5% FBS. Shown are flow cytometry plots compared to recovery in medium. The virus titer of No.3251 (pAAV.MND.mCherry.FKBP.IL2RG) was 6.55×10 ¹⁰ and that of No.3207 (pAAV.MND.GFP.FRB.IL2RB) was 2.50×10 ¹² . there were. The amount of each AAV virus used in each editing reaction was 10% of the medium volume. Panel B shows histograms showing the percentage of double-negative, GFP-positive, mCherry-positive and GFP/mCherry double-positive cell populations in double-edited cells.

Double-edited cells that have edited two loci are highly enriched by selection by treatment with rapalogs. A double-editing method for editing two loci was performed by the method shown in FIG. Panel A shows the expression of GFP and mCherry in mock-edited cells and GFP/mCherry (No.3207/No.3251)-edited cells (edited in medium containing 2.5% serum). Shown are flow cytometry plots comparing 10 days of treatment with 2 versus 10 days of treatment with 100 nM rapalog (AP21967). Panel B shows the percentages of double-negative, GFP-positive, mCherry-positive and GFP/mCherry double-positive cells in the double-edited cell population after 10 days of treatment with IL-2 and 10 days with rapalog. Histograms compared with treatment are shown.

It relates to a double-editing method that edits two loci of human CD4+ T cells. The timeline of the double editing process of CD4+ T cells using AAV and the expansion culture process using rapalog, and each editing condition are shown. Using human TRAC gRNA_4 and human FOXP3 gRNA_T9 with AAV No.3251 (MND.mCherry.FKBP.IL2RG) and AAV No.3207 (MND.GFP.FRB.IL2RB) (double editing of two loci) Editing method), gene editing of human CD4+ T cells was performed. Editing conditions were varied as described in the table by using different proportions of virus stock in the presence or absence of HDR enhancer or DMSO. Immediately after electroporation, cells were seeded in medium containing 2.5% FBS (recovery medium). Approximately 16 hours later, the medium was changed to medium containing 20% FBS and FACS analysis was performed on day 3 to determine the editing rate. Cells recovered in 2.5% FBS-containing media were cultured in the presence of IL-2 or rapalogs for an additional 10 days to monitor enrichment.

FIG. 10 shows a graph demonstrating that using an optimized amount (10% by volume) of AAV HDR donor improves double-editing efficiency. Gene editing was performed by the method summarized in FIG. Double-negative cell population, GFP-positive cell population, mCherry in double-edited cells 3 days after editing with varying amounts of AAV No.3207 and AAV No.3251 in the presence of 30 μM HDR enhancer or DMSO The percentage of positive and GFP/mCherry double-positive cell populations is shown graphically.

Data showing that selection with rapalogs can be highly enriched for double-edited CD4+ T cells edited at two loci are shown. Optimal results were obtained using media containing 2.5% FBS and an optimized amount (10% by volume) of AAV donor. Gene editing was performed by the method summarized in FIG. Cells shown in FIG. 10 (gene-edited cells with optimized amount (10%) of virus in medium containing 2.5% serum in the presence or absence of HDR enhancer) were transfected with IL- Graphs shown as the percentage of double-negative, GFP-positive, mCherry-positive and GFP/mCherry double-positive cells in the edited cell population after exposure to 2 or rapalogs for 10 days.

Schematic representation of a typical split-type CISC construct for the insertion of islet-specific TCR and FOXP3 and enrichment of double-edited cells using the double-editing method of editing two loci. IL-2 CISC (chemoinducible signaling complex) is divided into two different constructs and co-expressed with T1D4 TCR (No.3243) or FOXP3 (No.3252). Each construct comprises a rapamycin-binding and heterodimerizing complex half (one of the FKBP or FRB domains) linked to a chimeric endoplasmic reticulum targeting domain, the FKBP or FRB domain is fused to one half of the IL-2R signaling complex (IL-2RB or IL-2RG), which consists of transmembrane and intracellular domains. By delivering cDNA encoding each CISC component co-expressed with the T1D4 TCR or FOXP3 to primary human CD4+ T cells, dual-edited cells containing both CISC components and thus expressing T1D4 TCR and FOXP3 were obtained. can only grow.

A typical method for dual editing of a single locus using the promoter of the TRAC locus is shown. Schematic representation of an AAV construct for gene editing using homologous recombination repair designed for double editing within the TRAC locus. The upper panel shows a schematic diagram of the AAV construct (No.3240) for the expression of FOXP3 and introduction of split CISCs, and the lower panel shows the T1D4 TCR and split CISCs using the endogenous promoter of the TRAC locus. Schematic representation of an AAV construct (No. 3258) that is knocked in-frame into exon 1 of the TRAC locus to induce expression.

We show that editing of the TRAC locus using homologous recombination repair disrupts the expression of the TCR and strongly expresses the transgene by the enhancer and promoter of the endogenous TRAC locus. Panel A shows the editing strategy for in-frame integration of the mCherry-split CISC cassette into the endogenous TRAC locus. Using gRNAs targeting exon 1 of the TRAC locus, a construct (No. 3253) containing a fluorochrome marker (mCherry) followed by a P2A element and followed by FRB-IL2RB CISC was generated in-frame. Integration drives expression by the promoter of the endogenous TRAC locus while disrupting expression of the endogenous TCR. FIG. B shows a timeline showing the steps of gene editing CD4+ T cells using AAV construct No.3253. Cells were stimulated with CD3/CD28 beads for 3 days prior to gene editing. Panel C shows flow cytometric analysis of CD3 and mCherry expression in cells 7 days after gene editing. Flow cytometry plots showed significant expression of mCherry with deletion of CD3 in edited cells compared to mock-edited controls and AAV-only controls.

A comparison of mCherry expression induced by the endogenous promoter of the TRAC locus and mCherry expression induced by the MND promoter is shown. Using another HDR donor (comparing No.3253 and No.3208), gene editing was performed in the same manner as in Fig. 67, and relative expression activity from the endogenous promoter at the TRAC locus and relative expression activity from the MND promoter The relative expression activity was evaluated and compared. Flow cytometry plots revealed that the expression level of mCherry induced by the endogenous promoter (using P2A.mCherry.FRB.IL2RB (No.3253)) ) was shown to be lower than that of mCherry induced by the MND promoter. The bottom figure shows data from replicate experiments performed with the No. 3253 donor.

Another exemplary double-editing strategy utilizing constructs targeted to the TRAC locus and/or the FOXP3 locus and knocked in-frame to utilize the endogenous promoter of the TRAC locus is shown. Schematic representation of a typical AAV donor construct for making antigen-specific airT cells selectable by IL-2 CISC for the purpose of testing the double-editing strategy for one locus or two loci. show. The T1D4 TCR is shown as a typical TCR and can be replaced with another TCR based on disease target or other relevant characteristics of therapeutic application. The same is true for the IL-2 DISC construct.

Double editing of human CD4+ T cells using a decoy-CISC (split DISC) construct. Figure A shows a schematic of an HDR knock-in construct (No. 3280) containing a split IL-2 DISC for selection of double-edited cells with rapamycin. To make decoy-CISCs (split-type DISCs), a free FRB domain to sequester rapamycin in the cytoplasm was added to the MND.mCherry.FKBP.IL2RG construct to give MND.mCherry.FKBP.IL2RG.FRB (No .3280). Each repair template (No. 3280 and No. 3207 (not shown)) is flanked by identical homologous arms matched to gRNAs targeting the TRAC locus. CD4+ T cells edited by integrating one copy of each construct are expected to be selectively expandable by treatment with rapalogs or rapamycin. Panel B shows a timeline showing the steps of dual editing of CD4+ T cells using AAV No. 3280 and AAV No. 3207, expansion in the presence of rapalog/rapamycin, and analysis of enriched cells. show. Cells were stimulated with CD3/CD28 beads for 3 days prior to gene editing. Two days after gene editing, cells were analyzed for GFP and mCherry expression by flow cytometry and cells were expanded in media containing 50 ng/ml human IL-2, 100 nM rapalogs or 10 nM rapamycin. Panel C shows a flow cytometry plot showing the percentage of GFP/mCherry double positive cells 3 days after gene editing.

Figure 2 shows that double editing of human CD4+ T cells with a split DISC construct yields rapamycin-selectable cells. Double editing was performed by the method shown in FIG. Panel A, GFP/mCherry double-positive cells after 8 days of culture in the presence of 50 ng/mL human IL-2, 100 nM rapalog (AP21967), 10 nM rapamycin, or no drug treatment. Flow cytometry plots showing the percentage of Panel B shows double-negative, GFP-positive, mCherry-positive and GFP/mCherry cells in double-edited cells after enrichment with IL-2, rapalog (AP21967) or rapamycin, or without drug treatment. Histograms showing the results of measuring the percentage of double-positive cells are shown.

A typical construct for testing double-edited (split DISC) Treg cells in vivo. Schematic representation of the FOXP3 split IL-2 DISC HDR knock-in construct (No. 3262) used in combination with the T1D4 CISC construct (No. 3243) for rapamycin selection of double edited cells. The CISC components are split between two constructs and co-expressed with HA-FoxP3 or T1D4 TCR. This FOXP3 CISC construct also contains the FRB domain and is predicted to protect mTOR signaling in the presence of rapamycin (FOXP3 DISC construct). Each repair template is flanked by identical homologous arms that match the gRNA targeting the TRAC locus. CD4+ T cells edited by integrating one copy of each construct would be capable of selective expansion regardless of whether treated with rapalog or rapamycin.

Additional schematics and data for the generation of mouse airT cells and their characterization are shown.

The repair template used for editing mouse Foxp3 is shown. Schematic diagrams of the different AAV.GFP.KI and AAV.LNGFR.P2A constructs generated are shown. Editing efficiency, FOXP3 expression and suppressive function by these constructs were tested in mouse T cells.

FIG. 4 shows a schematic showing the method used to generate mouse airT cells using the MND.GFP.KI HDR donor construct (or another HDR donor construct).

Concerning the production and enrichment of mouse airT cells expressing endogenous Foxp3 using another promoter. mock edited cells, cells edited with MND.GFP.KI (No.1331), cells edited with MND.LNGFR.P2A (No.3189 or No.3261), edited with PGK.LNGFR.P2A (No.3227) Fig. 3 shows flow cytometry plots showing LNGFR and GFP expression before and after enrichment of LNGFR by FACS sorting of cells edited with EF-1a-LNGFR.P2A (No. 3229). The top plot shows the initial edit rate and the bottom plot shows the enrichment after FACS sorting. These data indicated that each candidate donor construct could be used to generate airT cells.

Foxp3 expression levels in mouse airT cells using different homologous donor cassettes. Panel A shows flow cytometry plots showing the expression of LNGFR and GFP in gene-edited spleen-derived T cells using homologous recombination repair. Each plot contains unmanipulated control cells from C57BL/6 mice; mock-edited T cells, edited with MND.GFP.KI (No.1331), UCOE from C57BL/6 mice. T cells edited with MND.GFP.KI (No.3213) and T cells edited with PGK.GFP.KI (No.3209) are shown. FIG. B shows a histogram of the flow cytometry data of the expression level of FOXP3 shown in FIG. FIG. C shows a bar graph showing the MFI of FOXP3 in edTreg/air T cells and nTreg cells generated using various alternative HDR donor constructs shown in the graph. FOXP3 expression was highest in donors containing the MND promoter.

Design and results of in vitro suppression assays using mouse tTreg cells or mouse airT cells are shown. A) For use in in vitro suppression assays, airT cells were enriched by FACS sorting 2 days after gene editing and resuspended in RPMI medium containing 10% FBS. nTreg cells (CD4+CD25+), T _eff cells (CD4+CD25-) and antigen-presenting cells (APCs) were isolated by column enrichment from a mixture of spleen cells and lymph node cells from 8-10 week old C56BL/6 mice. ) (CD4-CD25-) was isolated. Enriched 5×10 ⁶ T _eff cells were resuspended in 2 ml PBS, labeled by adding Cell Trace Violet (CTV) and incubating at 37° C. for 15 minutes, washed with medium, and placed in medium. Resuspend prior to addition to inhibition assays. 1.25×10 ⁵ APCs irradiated (2500 rad), 0.25×10 ⁵ T _eff cells, and nTreg or airT cells counted in a U-bottom 96-well tissue culture plate in a total volume of 300 μl medium. were co-cultured in the presence of 1 mg/ml anti-CD3 and incubated for 4 days in a 37° C. CO ₂ incubator. On day 4, cells were washed twice with PBS, stained with live/dead indicators, anti-CD4, anti-CD45 and anti-CD25 antibodies, and the inhibitory effect of airT cells on T _eff cell proliferation was examined by FACS (Fig. LSRII). B) Representative flow cytometry data showing that T _eff cell proliferation was suppressed in the presence of airT cells.

Fig. 2 shows the results of an in vitro test of the suppressive function of mouse airT cells. In the presence of mock-edited T cells, MND.GFP.KI (No.1331)-edited T cells, MND.LNGFR.P2A (No.3261)-edited T cells or nTreg cells from C57BL/6 mice, or Flow cytometry plots showing Cell Trace Violet labeled CD4+ T cells in the absence. These data indicate that mouse airT cells (generated using MND.GFP.KI HDR donors or MND.LNGFR.P2A HDR donors) and nTreg cells both exhibited potent suppressive function in vitro, and that suppressive function was shown to be similar.

Fig. 2 shows the in vitro suppressive function of mouse airT cells with various alternative promoters. C57BL/6 mouse-derived mock-edited T cells, T cells edited with MND.GFP.KI (No.1331), T cells edited with MND.LNGFR.P2A (No.3261), PGK.LNGFR.P2A ( Flow showing Cell Trace Violet-labeled CD4+ T cells in the presence or absence of T cells edited with No. 3227), T cells edited with EF-1a.LNGFR.P2A (No. 3229), or nTreg cells Cytometry plots are shown. Mouse airT cells with the MND promoter exhibit similar suppressive function to nTreg cells. In contrast, airT cells with the PGK promoter and airT cells with the EF-1α promoter exhibit only limited or no repressive function.

Fig. 2 shows the experimental design comparing LNGFR+ edited cells enriched by FACS sorting with LNGFR+ edited cells enriched by column purification in the NSG adoptive transfer model. The number of recipient NSG host animals in the five experimental groups and the origin and number of adoptively transferred control, airT or nTreg cells are shown in the table.

Flow cytometric analysis before and after column purification of NOD BDC2.5+ mouse cells edited with LNGFR.P2A. Figure A shows flow cytometry plots showing the expression of LNGFR in mock-edited cells and cells edited with MND.LNGFR.P2A (No.3189). Panel B shows flow cytometry plots showing LNGFR expression in MND.LNGFR.P2A (No.3189) edited cells enriched by FACS sorting. FACS sorting consistently enriched the edTreg cell products to greater than 90% purity, which was used to perform in vitro and in vivo studies.

Results of flow cytometric analysis of gene-edited mouse cells before and after column enrichment are shown. Flow cytometry plots before and after column enrichment of cells edited with MND-LNGFR.P2A (No.3189) are shown. In this enrichment example, 72 x ¹⁰⁶ cells with an initial editing rate of ~7% were added to the anti-LNGFR column, yielding 2 x ¹⁰⁶ edTreg cells with >84% purity. .

We present the experimental design and results to evaluate the function of islet antigen-specific airT cells in the NSG adoptive transfer model. A comparison of FACS-sorted airT cells and column-enriched airT cells is shown. 8-10 weeks of age by delivery of islet antigen-specific (BDC) airT cells (generated using HDR donors (3261 or 3389)) or antigen-specific nTreg cells into the retroorbital plexus (RO) were adoptively transferred into adult NSG recipient mice and then injected with antigen-specific T _eff cells. Mice were monitored for development of diabetes for up to 60 days. Diabetes after administration of the indicated mock-edited cells, MND.LNGFR.P2A-edited cells (FACS sorted or column-enriched) or nTreg and effector cells from NOD BDC2.5 mice. 1 shows a graph showing the percentage of mice that developed . Column-enriched antigen-specific MND.LNGFR.P2A airT cells reduced the development of diabetes in NSG mice and exhibited comparable function to FACS-sorted airT cells. High doses of column-enriched MND.LNGFR.P2A airT cells or nTreg cells completely protected recipient mice from developing diabetes.

Shown are the results comparing the in vivo function of airT cells generated using different promoters in the NSG adoptive transfer model. Genetically engineered antigen (BDC)-specific airT cells (airT cells generated using the MND promoter (donor construct 1331) or airT cells generated using the PGK promoter (donor construct 3209)) or antigen-specific nTreg cells were adoptively transferred into NSG recipient mice and then injected with antigen-specific T _eff cells. Mice were monitored for development of diabetes for up to 60 days. Mock-edited cells, MND.GFP.KI (No.1331)-edited airT cells, PGK.GFP.KI (No.3209)-edited airT cells or nTreg cells from NOD BDC2.5 mice as indicated in the graph. (5×10 ⁴ each) and effector cells (5×10 ⁴ ), showing the percentage of mice that developed diabetes. Antigen-specific airT cells with the MND promoter prevented the development of diabetes in all recipient mice. nTreg cells prevented diabetes in 4 of 5 recipient mice. In contrast, little or no protective effect was observed in antigen-specific airT cells that incorporated the PGK promoter. These data directly demonstrate that protection from type 1 diabetes is specific to airT cells generated using the MND promoter to induce expression of Foxp3, suggesting that type 1 diabetes or The feasibility of using this construct in human clinical trials for other immune diseases was supported.

Figure 4 shows that islet antigen-specific MND.GFP.KI airT cells remain viable in vivo for at least 60 days in the target organ (pancreas) and display a stable phenotype. Flow cytometry plots show FOXP3 and GFP expression in MND.GFP.KI (No.1331) NOD BDC2.5 airT cells harvested from the pancreas 60 days after adoptive transfer into NSG mice. Data show results from two mice. Recipient mice show proliferation of iTreg cells (FOXP3+GFP-CD4 T cells) or endogenous Treg cells derived from the transferred T _eff cell population (possibly a beneficial bystander effect of airT cell delivery). It seems to have happened secondarily due to

Design and results of targeting mouse Rosa26 locus with CRISPR for knock-in/knock-out. Panel A shows that the Rosa26 locus was chosen as a model safe harbor site for integration into mouse T cells by homologous recombination repair. Locations of two novel gRNAs (gRNA_1 and gRNA_2) within the mouse Rosa26 locus are shown. The gRNAs reported by Pesch et al. and the gRNAs reported by Wu et al. include previously reported gRNAs within this locus region. Panel B shows on-target site-specific activity as measured by ICE (Inference of CRISPR Edits), which indicates that Rosa26 was activated by delivering Cas9-RNP to primary mouse CD4 T cells using R26 gRNA_1. It was shown that locus-specific indels were induced.

A summary of gene editing experiments at the mouse Rosa26 locus using homologous recombination repair is shown. Figure A shows a schematic of AAV construct No. 3245 used in this experiment. Gene editing of mouse T cells using homologous recombination repair induces GFP expression by the MND promoter. Figure B shows the timeline of the experimental procedure. Mouse C57BL/6J CD4+ T cells were isolated and stimulated with CD3/CD28 beads for 3 days prior to gene editing. Expression of GFP in cells was assessed by flow cytometry on days 3 and 8 after gene editing.

Data showing that gene editing using homologous recombination repair was performed within the Rosa26 locus of mouse CD4+ T cells. CD4+ T cells were gene edited according to the method outlined in Figure 88 and evaluated by flow cytometry on day 3. Figure A shows flow cytometry plots showing the expression of GFP in mock-edited cells, cells edited with AAV No.3245 alone, and cells edited with AAV No.3245 and RNP 3 days after gene editing. Panel B shows histograms showing viability, percentage of GFP-positive cells and percentage of GFP-high expressing cells in gene-edited cell populations.

FIG. 4 shows that stable GFP expression is maintained in Rosa26 locus-edited mouse T cells using homologous recombination repair. CD4+ T cells were gene edited according to the method outlined in Figure 88 and evaluated by flow cytometry on day 8. Figure A shows flow cytometry plots showing the expression of GFP in mock-edited cells, cells edited with AAV No.3245 alone, and cells edited with AAV No.3245 and RNP 8 days after gene editing. Panel B shows a histogram showing the percentage of GFP-positive cells in the gene-edited cell population.

Schematic representation of AAV HDR donor constructs for expressing LNGFR linked to mouse Foxp3 and P2A within the Rosa26 locus of mouse T cells. These repair templates are flanked by 300-bp homologous arms adapted to the R26_gRNA_1 cleavage site and contain different promoters (MND or PGK) that drive the expression of mFOXP3 and LNGFR. In addition, a cassette containing the 4×CDK phosphorylation site mutant of Foxp3 is also included, and it is predicted that this construct can improve the stability of Foxp3.

Lentiviral CISC constructs used to transduce mouse CD4+ T cells and test selective expansion with rapalogs. Figure A shows a schematic diagram of lentiviral construct No.1272. This construct was developed to demonstrate the concept that human CISC components can be utilized to enrich mouse T cells in the presence of rapalogs. Upon transduction of mouse T cells, the MND promoter drives expression of mCherry linked to IL-2 CISC components (FKBP-IL2RG and FRB-IL2RB). Figure B shows the timeline of the experimental procedure. Mouse C57BL/6J CD4+ T cells were stimulated with CD3/CD28 beads for 3 days prior to transduction. Expression of mCherry in cells was assessed by flow cytometry on days 2 and 5 after transduction.

Mouse CD4+ T cells transduced with lentivirus containing CISC are highly enriched in the presence of rapalogs. Figure A shows flow cytometry plots showing expression of mCherry 2 days after mock transduction of mouse CD4+ T cells or transduction with lentivirus (No. 1272). Panel B. Flow cytometry plot showing expression of mCherry when mock mouse cells were treated with IL-2, IL-7 and IL-15 or mice transduced with lentivirus (#1272). Flow cytometry plots showing expression of mCherry when cells were treated with IL-2, IL-7 and IL-15, rapalog alone, or rapalog plus bead stimulation are shown.

AirT cells suppress proliferation of CD8+ T cells and CD4+ T cells.

FIG. 1 shows a schematic diagram of a method for generating antigen-specific airT cells by stimulating with a model antigen peptide (MP) and expressing FoxP3 by gene editing.

Antigen-specific suppression by airT cells specific for model antigenic peptides (MPs) is shown. They are briefly described below. T _eff : day 23 T cells stimulated with MP peptide (right) or HA peptide (left). Treg: Day 23 gene-edited cells specific for MP peptide (right) or HA peptide (left) edited with AAV containing CRISPR/Cas9 and Foxp3-MND-LNGFRki. APC: irradiated CD4-CD25+ autologous cells. Incubated for 6 days in the presence of DMSO or 5 μg/ml HA peptide.

Figure 2 shows that airT cells exert suppressive activity on the proliferation of T _eff cells. They are briefly described below. Both were incubated for 3 days. For bead-stimulated suppression assays, T _eff cells and Treg cells (untransduced (untd) edTreg cells, T1D5-1 airT cells or T1D5-1 mock cells) were co-cultured. For antigen-specific suppression assays, T1D5-1 T _eff cells and Treg cells were co-cultured. Gates for T _eff cells were set to CD4+CD11c-CTV+EF670-mTCRb+.

AirT cells suppress cytokine production by T _eff cells. They are briefly described below. T1D4 T _eff cells and (day 10) Treg cells (T1D4 mock cells or T1D4 airT cells) were incubated for 3 days in the presence of 1 μg/ml peptide.

We show that airT cells exert antigen-specific suppressive and bystander suppressive effects on T _eff cells. They are briefly described below. 1.25×10 ⁴ T _eff cells, 2.5×10 ⁴ Treg cells and 1×10 ⁵ APC were co-cultured in the presence of 5 μg/ml peptide.

We show that airT cells exert antigen-specific suppressive and bystander suppressive effects on T _eff cells. They are briefly described below. 1.25×10 ⁴ T _eff cells, 2.5×10 ⁴ Treg cells and 1×10 ⁵ APC were co-cultured in the presence of 5 μg/ml peptide.

Fig. 2 shows bystander inhibitory effects on cytokine production by T _eff cells. They are briefly described below. T1D5-2 T _eff cells and (day 10) Treg cells (T1D4 mock cells or T1D4 edTreg cells) were incubated for 3 days in the presence of 1 μg/ml peptide.

Proliferation assays to confirm TCR dose response are shown. They are briefly described below. mTCR expression data were obtained 8 days after transduction. Proliferation assays were performed using day 11 cells and incubated for 4 days.

Fig. 3 shows an mTCRβ expression assay and a proliferation assay for verifying the expression of TCRs specific to pancreatic islet antigens. They are briefly described below. T cells: Cells 9 days after transduction labeled with Cell Trace Violet. APC: irradiated CD4-CD25+ cells. Incubated for 5 days.

Detectable antigen-specific GFP+airT cells in the pancreas. See also FIGS. 107 and 116.

Generation and enrichment of murine LNGFR+airT cells used for in vivo suppression studies. See also FIG.

Antigen-specific MND.LNGFR.P2A-air T cells completely prevented diabetes in NSG mice. See also FIGS. 115, 134 and 135.

Detectable antigen-specific GFP+airT cells in the pancreas.

Schematic representation of a typical IL-2 CISC according to the present disclosure and data associated with this IL-2 CISC.

FIG. 4 shows that contact with rapamycin in vivo improves CISC cell persistence.

1 shows a schematic representation of a typical gene-edited cell according to the present disclosure. FIG.

Concerning the selection of gRNAs targeting the TRAC locus.

A double-editing strategy using a split IL-2 CISC component targeting the TRAC locus.

Shows selection of double editing cells by assembly of CISC components in vitro.

Flow cytometric analysis before and after column purification of NOD BDC2.5+ mouse cells edited with LNGFR.P2A. Figure A shows flow cytometry plots showing specific LNGFR expression in cells edited with MND.LNGFR.P2A (No.3261), but not in mock cells. . Panel B is a flow cytometry plot showing the flow-through fraction (F.T.) from column purification of cells edited with MND.LNGFR.P2A (No.3261) and the expression of LNGFR in the eluted sample after concentration. show. Column enrichment yielded an airT cell product with a purity of 74.5% for in vivo studies.

Figure 3 shows functional assessment of islet antigen-specific airT cells in an NSG adoptive transfer model. Delivery of islet antigen-specific (BDC) airT cells (generated using HDR donor 3261) or antigen-specific nTreg cells (50,000 each) into the retroorbital venous plexus (RO) resulted in 8-10 Age-old adult NSG recipient mice were adoptively transferred and then injected with 50,000 antigen-specific T _eff cells. Panel A shows the CD4 and CD25 expression profiles in nTreg cells and flow cytometry plots showing the expression of LNGFR+ in cells edited with MND.LNGFR.P2A (No.3261). FIG. B shows a graph showing the results of monitoring the development of diabetes in mice for up to 49 days. Percentage of mice that developed diabetes following administration of the indicated mock-edited cells, MND.LNGFR.P2A-edited cells (enriched in columns) or nTreg and effector cells from NOD BDC2.5 mice. shows a graph showing Column-enriched antigen-specific MND.LNGFR.P2A-air T cells completely prevented diabetes in NSG mice.

Figure 4 shows that islet antigen-specific MND.GFP.KI airT cells remain viable in vivo for at least 49 days in the target organ (pancreas) and display a stable phenotype. Flow cytometry plots show LNGFR and FOXP3 expression in NOD BDC2.5 airT cells harvested from the pancreas 49 days after adoptive transfer into NSG mice.

Flow cytometry plots analyzing mTCRb expression gating on CD4+ cells 9 days post-transduction are shown.

CD4+ T cells transduced with a TCR specific for rheumatoid arthritis antigen were labeled with CTV and co-cultured with APCs (irradiated PBMCs) for 3 days in the presence of peptides recognized by the TCR or DMSO. Shown are the indicated flow cytometry plots.

Polyclonal and antigen-specific suppression assays using enolase-specific edTreg cells are shown.

The percentage growth inhibition of Teff cells by no Treg cells, untd edTreg cells, Enol edTreg cells or mock cells in the presence of anti-CD3/CD28 (black) or in the presence of APC and enolase peptide (grey) is shown in Figure 118B. FIG. 10 shows a graph showing results calculated from the proliferation rate obtained.

Non-transduced edTreg cells or CILP297 by gating on LNGFR+Foxp3+ in edited cells that were not lentivirally transduced or transduced with lentivirus encoding CILP297-1-TCR. Flow cytometry plots analyzing mTCRb expression in -1 edTreg cells are shown.

Polyclonal and antigen-specific suppression assays using CILP-specific edTreg cells are shown.

The growth inhibition rate of CILP Teff cells by no Treg cells, untd edTreg cells, CILP edTreg cells or mock cells in the presence of anti-CD3/CD28 (black) or APC and CILP peptide (grey) is shown in Figure 119B. Graphs showing results calculated from the indicated proliferation rates are shown.

Non-transduced edTreg cells or Vim418 edTreg cells by gating with LNGFR+Foxp3+ on edited cells that were not lentivirally transduced or transduced with lentivirus encoding Vim418-TCR. Flow cytometry plots analyzing the expression of mTCRb in .

Polyclonal and antigen-specific suppression assays using vimentin-specific edTreg cells are shown.

The growth inhibition rate of Vim Teff cells by no Treg cells, untd edTreg cells, Vim edTreg cells or mock cells in the presence of anti-CD3/CD28 (black) or in the presence of APC and vimentin peptide (grey) is shown in Figure 120B. Graphs showing results calculated from the indicated proliferation rates are shown.

Edited cells not transduced with lentivirus, edited cells transduced with lentivirus encoding Agg520-TCR or edited cells transduced with lentivirus encoding Vim418-TCR Flow cytometry plots analyzing mTCRb expression in non-transduced edTreg, Agg520 edTreg or Vim418 edTreg cells by gating on LNGFR+Foxp3+ are shown.

Polyclonal suppression assay using Agg520 or Vim418 specific edTreg or mock cells and Agg520 Teff cells.

FIG. 121B shows a graph showing the results of inhibition of growth of Agg520 Teff cells by no Treg cells, untd edTreg cells, Agg edTreg cells, Agg mock cells, Vim edTreg cells, or Vim mock cells, calculated from the growth rates shown in FIG. 121B. .

Antigen-specific and bystander suppression assays using Agg520 or Vim418-specific edTreg or mock cells and Agg520 Teff cells are shown.

FIG. 121D shows a graph showing the results of calculating the growth inhibition rate of Agg520 Teff cells by no Treg cells, edTreg cells, or mock cells from the growth rates shown in FIG. 121D.

Edited cells not transduced with lentivirus, edited cells transduced with lentivirus encoding CILP297-1-TCR or edited with lentivirus encoding Vim418-TCR Flow cytometry plots analyzing mTCRb expression in non-transduced edTreg, CILP297-1 edTreg or Vim418 edTreg cells by gating the cells with LNGFR+Foxp3+ are shown.

Polyclonal suppression assay using CILP297- or Vim418-specific edTreg or mock cells and CILP297-1 Teff cells.

FIG. 122B shows a graph showing the results of suppressing proliferation of CILP Teff cells by no Treg cells, untd edTreg cells, CILP edTreg cells, CILP mock cells, Vim edTreg cells, or Vim mock cells, calculated from the proliferation rates shown in FIG. 122B. .

Antigen-specific and bystander suppression assays using CILP297- or Vim418-specific edTreg or mock cells and CILP297-1 Teff cells are shown.

FIG. 122D shows a graph showing the results of calculating the growth inhibition rate of CILP Teff cells by no Treg cells, edTreg cells, or mock cells from the growth rate shown in FIG. 122D.

Flow cytometry plots showing expression of mTCRb and LNGFR/Foxp3 at day 7 in edited cells expressing SLE3-TCR are shown.

Polyclonal and antigen-specific suppression assays using SLE-specific edTreg cells are shown.

Schematic representation of an AAV HDR donor construct designed to introduce each component of the split-type CISC into the TRAC locus using the single locus double-editing method.

The timeline of the dual editing process of CD4+ T cells with AAV and the expansion culture process in the presence of rapalogs, which are important in this study, are shown.

Flow cytometry showing expression of T1D4 and FOXP3 in mock-edited, single-edited and double-edited cells (using 10% by volume of AAV No.3243 and AAV No.3240, respectively) 3 days after gene editing. Show the plot.

Flow cytometry plots showing expression of T1D4 and CD4 in mock-edited cells and cells edited with the virus mixture are shown.

Histograms showing the percentage of double-negative, FOXP3-HA-positive, T1D4-positive, and FOXP3/T1D4 double-positive cells in double-edited cells are shown.

Histograms comparing CD3 knockout rates in FOXP3/T1D4 double-edited cells and mock-edited cells are shown.

Flow cytometry plots showing viability and expression of T1D4 and FOXP3 after treatment of double-edited cells with 50 ng/mL IL-2 (top) or 100 nM rapalog (AP21967; bottom) for 7 days.

Flow chart comparing CTLA4 expression in T1D4/FOXP3 double-positive and double-negative cell populations after treatment with 50 ng/mL IL-2 (top) or 100 nM rapalog (AP21967; bottom) for 7 days. Cytometry plots are shown.

Double-edited cells treated with 50 ng/mL IL-2 (upper plot) and double-edited cells treated with 100 nM AP21967 followed by recovery in medium containing IL-2 (lower plot). Flow cytometry plots comparing survival (right plot) and expression of T1D4 and FOXP3 (left plot) are shown in .

Enrichment over 10 days when T1D4/FOXP3 double-positive cells treated with 50 ng/mL IL-2 or 100 nM rapalog (AP21967) were allowed to recover in recovery medium containing IL-2 for the last 3 days. A graph showing magnification is shown.

Schematic representation of HDR knock-in construct (No. 3280) containing split IL-2 DISC for selection of double-edited cells with rapamycin or rapalog.

The timeline of the double-editing step of CD4+ T cells using AAV No.3280 and AAV No.3207, expansion culture step in the presence of rapalog/rapamycin, and analysis step of enriched cells, which are important in this study, are shown below. show.

Flow cytometry plots showing the expression of mCherry and GFP in double-edited cells (AAV donor No. 3280 and AAV donor No. 3207 added at 10% of medium volume each) 4 days after gene editing. The virus titer of No.3280 was 3.30×10 ¹² and the virus titer of No.3207 was 3.1×10 ¹⁰ . The initial double-positive rate across 4 million double-edited cells was 4.47%. The total number of cells seeded in the gRex flask was 7.6 million, of which 340,000 were double positive cells.

Flow cytometry plots showing viability (upper panel) and GFP and mCherry expression (lower panel) after 7.6 million edited cells were seeded in gREX flasks and expanded in the presence of AP21967 for 7 days. A 32-fold increase in double-positive cells was shown. The total number of double positive cells in the gRex flask was 11.1 million.

Figure 3 shows the timeline of the dual editing steps of CD4+ T cells using AAV No.3280 and AAV No.3207, the expansion steps in the presence of rapalogs, and the analysis steps of the enriched cells.

Flow cytometry plots showing expression of mCherry and GFP in double-edited cells (AAV No.3280 and AAV No.3207 added at 10% each) are shown. No.3280 (MND.mCherry.FKBP.IL2RG.FRB) had a viral titer of 3.30×10 ¹² and No.3207 (pAAV.MND.GFP.FRB.IL2RB) had a viral titer of 3.1×10 ¹⁰ . there were. A total of 10 million cells were gene-edited with an initial double-positive rate of 2.37%. A gRex flask was seeded with 9.1 million cells, of which 216,000 were double positive cells.

Flow cytometry plots showing viability and GFP and mCherry expression after gene-edited cells were seeded in gREX flasks and expanded in the presence of AP21967 for 7 days. After 7 days of expansion culture, the total number of double-positive cells in the gRex flask was 9.7 million, representing an approximately 45-fold increase from the initial seeding of 216,000 double-positive cells. shown.

Design of in vitro suppression assays using mouse edTreg cells or mouse nTreg cells.

Representative flow cytometry data showing decreased proliferation of BDC2.5+ Teff cells in the presence of BDC2.5+ edTreg cells.

Flow showing Cell Trace Violet-labeled CD4+ T cells in the presence or absence of mock cells, MND.LNGFR.p2A (No.3261)-edited Treg cells or nTreg cells from NOD BDC2.5+ mice. Cytometry plots are shown. Islet antigen-specific TCR+ mouse edT _reg cells (generated using MND.LNGFR p2A (No.3261) HDR donor) and mouse tTreg cells exhibited antigen-specific suppressive function in vitro. 50,000 Teff cells + anti-CD3 (1 μg/ml) + 200,000 irradiated (2500 rad) APCs. Analyzed on day 4. CTV = Cell Trace Violet. Data from experiments performed with a 1:1 ratio of Treg to Teff are shown.

FIG. 10 shows a graph showing the percentage of mice that developed diabetes after administration of the indicated mock-edited cells, MND.LNGFR.P2A-edited cells or nTreg cells and effector cells from NOD BDC2.5 mice. MND.LNGFR p2A edTreg cells, like nTreg cells, completely prevented diabetes development, whereas mock-edited control cells had no effect on diabetes development.

Percentage of mice that developed diabetes after administration of the indicated mock-edited cells, MND.LNGFR.P2A-edited cells or nTreg cells plus effector cells from NOD BDC2.5 mice in replicate experiments. Show the graph. Column-enriched antigen-specific LNGFR p2A edTreg cells completely prevented diabetes in NSG mice (day 33).

Figures 136A, 136B and 136C are lists of amino acid sequences of CDR3 and J regions of TCR α and β chains that specifically recognize antigens associated with the pathogenesis of autoimmune, allergic or inflammatory diseases. shows a table showing

Shown is a table listing the amino acid sequences of the CDR3 and J regions of the α and β chains of TCRs that specifically recognize antigens associated with the pathogenesis of autoimmune, allergic or inflammatory diseases.

A table listing the amino acid sequences of the J regions of TCRs that specifically recognize antigens associated with the pathogenesis of autoimmune, allergic or inflammatory diseases is presented.

FIG. 1 shows a table listing nucleotide sequences encoding TCR α and β chain V regions that specifically recognize antigens associated with the pathogenesis of autoimmune, allergic or inflammatory diseases.

A table listing the amino acid sequences of the J regions of TCRs that specifically recognize antigens associated with the pathogenesis of autoimmune, allergic or inflammatory diseases is presented.

FIG. 1 shows a table listing nucleotide sequences encoding TCR α and β chain V regions that specifically recognize antigens associated with the pathogenesis of autoimmune, allergic or inflammatory diseases.

A table listing the amino acid sequences of the J regions of the α and β chains of TCRs that specifically recognize antigens associated with the pathogenesis of autoimmune, allergic or inflammatory diseases is provided.

FIG. 2 shows a table listing amino acid sequences of antigenic epitopes recognized by TCRs specific for CYP2D6 antigens associated with hepatitis type 2 autoimmune.

FIG. 1 shows a table listing the amino acid sequences of antigenic epitopes recognized by TCRs specific for the BP230 or BP180 antigens associated with bullous pemphigoid.

Amino acid sequences of polypeptide antigens associated with the etiology of autoimmune, allergic and/or inflammatory diseases and containing antigenic epitopes recognized by specific TCRs, and the α-chains of TCRs that specifically recognize these antigens and a table listing the amino acid sequences of the CDR3 regions of the β chains.

Amino acid sequences of polypeptide antigens associated with the etiology of autoimmune, allergic and/or inflammatory diseases and containing antigenic epitopes recognized by specific TCRs, and the α-chains of TCRs that specifically recognize these antigens and a table listing the amino acid sequences of the CDR3 regions of the β chains.

Amino acid sequences of polypeptide antigens associated with the etiology of autoimmune, allergic or inflammatory diseases and containing antigenic epitopes recognized by specific TCRs, and the α and β chains of TCRs that specifically recognize these antigens Shown is a table listing the amino acid sequences of the CDR3 regions of the chains.

FIG. 2 shows a table listing specific nucleic acid sequences useful in embodiments provided herein, including guide RNAs (gRNAs) and AAV vectors containing FOXP3 editing sequences.

Some of the embodiments of the methods and compositions provided herein relate to antigen-specific artificial immunoregulatory T (airT) cells. The airT cells of the invention are also referred to as "edTreg" cells or "gene-edited Treg" cells. Some embodiments are artificial T cells (e.g., T lymphocytes) that express forkhead box protein 3/winged helix transcription factor (FOXP3) at levels equal to or greater than natural regulatory T (Treg) cells. CD4+CD25 with the FOXP3 gene artificially modified such that the gene product is constitutively expressed and at least one introduced polynucleotide encoding an antigen-specific T-cell receptor (TCR) polypeptide Including artificial T cells including + T cells.

In some embodiments, the airT cells of the invention are mediated by specific antigens recognized by the TCR, such as autoantigens, allergens, and other antigens associated with the pathogenesis of inflammatory diseases characterized by an exaggerated immune response. Induction can result in antigen-specific immunosuppression. Importantly, the production of the airT cells of the present invention requires the time associated with isolating the relatively rare (1-4% of human PBMC) natural Treg cells as starting material for gene editing. , without the associated costs and inefficiencies, the advantage of being able to generate therapeutically effective amounts of desired cells for the purpose of adoptive immunotherapy.

In some embodiments, the airT cells of the invention express functional TCRs that specifically recognize antigens associated with the pathogenesis of autoimmune, allergic or inflammatory diseases. Such functional TCRs include, for example, TCRs comprising any of the TCR polypeptide sequences disclosed herein, and TCR polypeptides encoded by the TCR-encoding polynucleotide sequences disclosed herein. and the like, including the TCR shown in the drawings.

In some embodiments, the airT cells of the invention express functional TCRs that specifically recognize antigens associated with the pathogenesis of autoimmune, allergic or inflammatory diseases. Such antigens include, for example, polypeptide autoantigens, allergens and/or inflammation-associated antigens, including the amino acid sequences of the polypeptide antigens disclosed herein, and amino acid sequences of the polypeptide antigens disclosed herein. Polypeptide autoantigens, polypeptide antigens that immunologically cross-react with allergens and/or inflammation-associated antigens, etc., including sequences, including antigens shown in the figures.

Certain embodiments disclosed herein relate to gene editing methods for making airT cells of the invention. In this gene editing method, (i) the expression of FoxP3 is induced by introducing a constitutive promoter into cells that do not express FoxP3, and FOXP3 is expressed at an expression level equal to or higher than that of natural regulatory T (Treg) cells. , stable expression of FoxP3 through targeted editing of the FoxP3 gene by maintaining a stable immunoregulatory (immunosuppressive) program regulated by FoxP3, and (ii) further gene editing of the same cells. and stably expressing an exogenous TCR by introducing a specific nucleotide sequence of the present disclosure that encodes a TCR that recognizes an antigen associated with the etiology of an autoimmune, allergic or inflammatory disease. Stable expression of FoxP3 and stable expression of an exogenous TCR via this targeted editing of the FoxP3 gene has a surprisingly favorable functional relationship, indicating that stable expression coupled with the expression of the desired TCR Recombinant T cells characterized by immunosuppressive capacity can be selected and expanded. While not wishing to be bound by any particular theory, some of the airT cell embodiments disclosed herein enhance the plasticity of natural Treg cells by stably expressing FoxP3 artificially. for indications where antigen-specific immunosuppression is desired, such as autoimmune diseases, allergic diseases or other inflammatory diseases, without the associated risks (e.g. conversion to T effector behavior) It is believed that safe and effective cells for adoptive transfer immunotherapy can be provided.

As an advantage of the method for producing airT cells disclosed in the present specification, as described above, if a normal production method is used, human peripheral blood mononuclear cells are present only at a low frequency in peripheral blood. A step of isolating natural Treg cells, of which only about 1-4% are found, is performed first, but in some embodiments of the invention, this isolation step is omitted. Alternatively, the airT cells of the invention can be generated by isolating CD4+ T cells, as described herein. CD4+ T cells include a wide variety of T cells compared to cells with other cell surface markers, but CD4+ T cells are present in relatively large numbers as they comprise approximately 25-60% of human PBMCs. It can be used as a starting material for gene editing by various methods provided herein.

In certain embodiments, compositions comprising antigen-specific immunoregulatory T (airT) cells of the invention and methods of producing airT cells of the invention are used to treat specific autoimmune, allergic and/or inflammatory diseases. can be used for the treatment and/or alleviation of (e.g., adoptive transfer immunotherapy) in which airT cells are stable and antigen-specific immunoregulatory function is enhanced. You can get unprecedented benefits from being maintained.

In certain embodiments, the airT cells described herein unexpectedly interact with antigens (e.g., any of the antigens disclosed herein) associated with the pathogenesis of autoimmune, allergic or inflammatory diseases. ) can induce an antigen-specific immunosuppressive reaction. Such antigen-specifically induced immunosuppression is
(i) suppression of one or both activation and proliferation of effector T cells that recognize an antigen specifically recognized by an airT cell TCR comprising said TCR polypeptide encoded by said at least one introduced polynucleotide; ,
(ii) expression of an inflammatory cytokine or inflammatory mediator by effector T cells that recognize an antigen specifically recognized by an airT cell TCR comprising said TCR polypeptide encoded by said at least one introduced polynucleotide; Suppression,
(iii) production of one or more immunosuppressive cytokines or anti-inflammatory products by airT cells, e.g. release of immunosuppressive cytokines or perforin/granzymes, induction of indoleamine-2,3-dioxygenase (IDO); construction of one or more inhibitory mechanisms, such as competition for IL2 or adenosine, tryptophan catabolism, expression of inhibitory receptors by airT cells, and (iv) said TCR polynucleotide encoded by said at least one introduced polynucleotide. One or more of suppression of activation and/or proliferation of effector T cells that do not recognize the antigen specifically recognized by the airT cell TCR containing the peptide may be included.
In some embodiments, such antigenic stimulation of airT cells is HLA-restricted.

In some embodiments, the generation of airT cells of the invention stably expressing FoxP3 as described herein can be found in conventional methodologies to express the FOXP3 transgene by retrovirus- or lentivirus-mediated gene transfer. It overcomes the shortcomings. Cell populations virally transduced with FoxP3 by such conventional methodologies are genetically heterogeneous cell populations, with this heterogeneity resulting in different genomic sites with different stabilities and different phenotypes. By random integration of the FOXP3 transgene at expression level. Such transduced cell populations exhibit, at least transiently, Treg characteristics, such as phenotypic markers and cytokine expression profiles, but are associated with the risk of genotoxicity and are localized to sites of viral integration. The risk of being susceptible to silencing by regulatory elements is likely to occur.

To avoid such risks, some of the embodiments provided herein utilize specific targeted gene editing to engineer the FOXP3 gene, rather than transferring the FOXP3 gene via a virus. change to Furthermore, gene editing may be performed by specifically targeting the TCR. In certain embodiments described herein, lentiviral-mediated gene delivery is used to introduce an autoimmune-associated TCR candidate into CD4 T cells, which are then transfected with the FOXP3 gene. Edit to stably force expression of FoxP3. In some related embodiments, this method is combined with gene editing methods that simultaneously delete (eg, delete by inactivation (also referred to herein as "knockout")) the endogenous TCR gene.

As an alternative to lentiviral-based delivery of TCRs, some of the embodiments described herein relate to simultaneous gene editing of separate alleles at the same locus, e.g. It relates to the double-editing method of editing two alleles at one locus by making two edits at one locus (using one guide RNA and multiple AAV donor homologous constructs).

As yet another method, some of the embodiments described herein relate to gene-editing two different loci simultaneously, e.g., using two different guide RNAs and It relates to the double-editing method of editing two loci by performing separate gene editing at each of the two loci (using an AAV donor homology cassette).

These methods may be used to deliver the recombinant FOXP3 gene and the recombinant TCR gene to one specific locus or two different specific loci. Further, as described herein, in some embodiments, the method includes the insertion of the TCR and Foxp3 genes by incorporating split-type chemo-inducible signaling complex (split-type CISC) components. selectively expanding only T cells that express both and enriching for airT cells.

chemo-inducible signaling complex (CISC)
As described herein, in some embodiments, methods utilizing split chemical-induced signaling complexes (CISCs) are used. In this method, (i) a FoxP3 gene product that is constitutively expressed by editing the FoxP3 gene and (ii) a heterologous TCR gene product by introducing an edited heterologous TCR gene are expressed in the same cell. Gene-edited airT cells may be obtained by the method, which may be selectively expanded. Expression of the constitutively expressed FoxP3 gene product by editing the FoxP3 gene is associated with cell surface expression of a first CISC component that specifically binds to a CISC-inducing molecule. The CISC component exists as a transmembrane fusion protein with an extracellular domain that binds a first CISC-inducing molecule, a transmembrane domain, and a first activation signaling intracellular domain. Expression of the heterologous TCR gene product by introduction of the edited heterologous TCR gene is associated with cell surface expression of a second CISC component, distinct from the first CISC component, that specifically binds to said CISC-inducing molecule. and the second CISC component comprises an extracellular domain that binds to a second CISC-inducing molecule, a transmembrane domain, and a second activation signaling intracellular domain separate from the first activation signaling intracellular domain. It exists as a transmembrane fusion protein with a signaling intracellular domain.

In certain embodiments, CD4+ T cells are obtained by enrichment from a biological sample, such as peripheral blood mononuclear cells (PBMC), prior to gene editing (eg, double editing) as described herein. In certain embodiments, CD4+ T cells obtained by enrichment are pretreated (e.g., solid-phase-immobilized anti-CD3 antibody and activated non-specifically with CD28 antibodies).

In some embodiments, exposing the dual-edited T cells described herein to a CISC-inducing molecule results in the extracellular domain of the first CISC component and the extracellular domain of the second CISC component. A CISC-inducing molecule binds to form a heterodimer consisting of a first CISC component and a second CISC component, the first activation signaling intracellular domain and the second activation signaling cell A functional signaling complex formed by the endodomain is activated. Thus, expression of the first CISC component and the second CISC component in the airT cell population results in the expression of FoxP3 and a heterologous TCR, allowing selective expansion of this airT cell population. becomes.

In some embodiments, a first CISC component is co-expressed with the FoxP3 gene product and a second CISC component is co-expressed with the TCR gene product, respectively, in the airT cells of the invention. One gene editing can be designed to occur at separate alleles of the same locus (e.g., double editing methods that edit two alleles) or can be designed to occur at two different loci. (eg, double-editing methods that edit two loci). In some embodiments, a third CISC component that specifically binds a CISC-inducing molecule may also be co-expressed with the FoxP3 or TCR gene product. This third CISC component, when expressed, remains intracellular and acts as a decoy to bind CISC-inducing molecules that can enter the interior of the cell, preventing toxicity by these CISC-inducing molecules.

Details of the CISC system, including the structure of the first CISC component, the structure of the second CISC component and the structure of the third CISC component, as well as the structure of the CISC-derived molecules, are set forth elsewhere herein, WO /2018/111834 and WO/2019/210078, both of which are expressly incorporated herein by reference in their entirety. Briefly, in WO/2018/111834, by knocking in (inserting) a genetic construct encoding a chemically-inducible signaling complex (CISC), a fusion protein capable of ligand-mediated dimerization, , describes compositions and methods for genetically editing host cells. By expressing the two subunits of this fusion protein in cells and then exposing the host cell to chemical ligands, dimerization of these subunits is induced to form CISCs and cell activation signals. can induce the transmission of Thus, by using the CISC system, it is possible to select and expand cells into which two CISC components have been genetically modified (for example, to induce proliferation by activation). Also, in WO/2019/210078, a nucleic acid sequence encoding a first CISC subunit component and a second Gene-editing compositions and methods for introducing into host cells nucleic acid sequences encoding CISC subunit components of are described. Induction of CISC subunit dimerization via chemical ligands induces biosignaling to allow selection and expansion of gene-edited cells. In some related embodiments, a nucleic acid encoding a third CISC subunit component may be expressed in a host cell. When expressed intracellularly, this third CISC subunit remains intracellular and can function as a decoy to suppress the deleterious effects of CISC ligands that have entered the cell interior. .

A typical first CISC subunit component and a second CISC subunit component each contain a functional intracellular signaling domain of the β subunit of the IL2 receptor (IL2RB) and a γ subunit of the IL2 receptor. Either one of the functional intracellular signaling domains of the unit (IL2RG) may be included. A typical third CISC component may include the rapamycin binding functional domain of the FK506 binding protein (FKBP).

Phenotypic markers and suppressor functions of FOXP3/airT cells
Editing the FOXP3 gene may involve artificial alteration of the native FOXP3 locus and/or may involve artificial alteration of a chromosomal site other than the native FOXP3 locus. For example, gene editing involves knocking in (e.g., inserting) a nucleic acid molecule comprising an exogenous FOXP3-encoding polynucleotide and a constitutive promoter operably linked thereto at a chromosomal site other than the native FOXP3 locus. Chromosomal sites other than the natural FOXP3 locus include, for example, T cell receptor α chain (TRAC) locus, T cell receptor β chain (TCRB) locus, adeno-associated virus integration site 1 (AAVS1), another locus, and so on. Thus, surprisingly, in certain embodiments, introduction of an artificial FoxP3 gene sequence at a genomic site other than the natural FOXP3 locus (e.g., the TRAC locus) results in antigen-specific immunosuppression, Also provided are airT cells that can constitutively express the FOXP3 gene product at an expression level equal to or higher than that of natural Treg cells.

In a specific embodiment, in the airT cells of the invention, two genes each giving rise to a FoxP3 gene product co-expressed with a first CISC component and a TCR gene product co-expressed with a second CISC component Gene editing may be designed to occur at separate alleles of the same locus (e.g., double-editing methods that edit two alleles) or may be designed to occur at two different loci ( For example, the double-editing method, which edits two loci). In some embodiments, surprisingly, the airT cells disclosed herein are effective for at least 21 days in vitro, or in vivo, in immune-compatible mammals requiring antigen-specific immunosuppression. Capable of expressing the FOXP3 gene product at levels sufficient to maintain the CD4+CD25+ phenotype for at least 60 days after adoptive transfer into an animal host and pathogenesis of an autoimmune, allergic or inflammatory disease or a TCR specifically recognizing an antigen described herein associated with the etiology of an autoimmune, allergic or inflammatory disease. can be expressed

Accordingly, in certain embodiments, the CD4+CD25+air T cells disclosed herein relate to genetically modified cells obtained by artificially modifying the FOXP3 gene of CD4+CD25- T cells. In some embodiments, the airT cells of the present invention constitutively express the FOXP3 gene product at an expression level equal to or higher than that of natural regulatory T (Treg) cells due to the aforementioned artificial modification. In some embodiments, the airT cells of the invention further express CD25, CD152 and/or ICOS markers on the cell surface at expression levels characteristic of immunoregulatory cells such as naive Treg cells. may However, in some embodiments, the airT cells of the present invention may exhibit a HeliosLo phenotype on the cell surface, unlike natural Treg cells, e.g. The Helios marker may be expressed on the cell surface at a scientifically significant low expression level.

Detailed examples of gene editing methods for inducing FoxP3 expression in T cells are described elsewhere in this specification, and are also described in WO/2018/080541 and WO/2019/210078 (both of which are are also expressly incorporated herein by reference in their entireties). Detailed examples of forced expression of FOXP3 using gene editing, including knock-in (insertion) of full-length codon-optimized FoxP3 cDNA into the FOXP3 or AAVS1 loci, are described in WO/2019/210042. (which document is expressly incorporated herein by reference in its entirety).

Briefly, WO/2018/080541 uses gene editing with Cas9, ZFNs or TALENs to knock in (e.g. insert) a constitutive promoter (EF1α promoter, PGK promoter or MND promoter). , describe CD4+ T cells stably expressing endogenous FoxP3. FoxP3 may be expressed by targeted knock-in (insertion) into the FOXP3 locus of a polynucleotide comprising sequences encoding exons of FOXP3 to be expressed first and regulatory sequences operably linked thereto. Said regulatory sequence may comprise a promoter, which in some embodiments is the MND promoter, the PGK promoter or the EF1α promoter, or another inducible promoter, another weak promoter or another constitutive promoter. There may be. Gene-edited typical FOXP3+ cells contain the FOXP3 gene with a fully methylated intronic regulatory T-cell-specific demethylated region (TSDR) upstream of the promoter-integrated knock-in site. good.

WO/2019/210078 describes the forced expression of FoxP3 in CD4+ T cells to obtain cells with a Treg-like phenotype, a method of selecting such cells with a Treg-like phenotype to obtain a Treg-enriched preparation, and methods for in vitro expansion of cell populations with such Treg-like phenotypes. This WO/2019/210078 also describes compositions and methods for editing target genes at the FOXP3 locus, the AAVS1 locus and/or the TCRα (TRAC) locus, which compositions and methods include contains guide RNA (gRNA) sequences specific for each of the FOXP3, AAVS1 and/or TCRα (TRAC) loci, and donor templates for gene editing via homologous recombination repair. Also, in WO/2019/210078, an activation signal for T cell proliferation is induced by dimerizing a first CISC component and a second CISC component via a chemical ligand. A CISC system has also been described that allows selective expansion of gene-edited T cells. Further, this WO/2019/210078 discloses that the CISC-inducing molecule is rapamycin or any of the various rapamycin analogues, rapamycin derivatives and rapamycin mimetics disclosed in this document, wherein activation of CISC components The signaling domain is located in the cytoplasm of the IL-2 receptor (IL2R), the IL-2 receptor beta subunit (IL2Rb (also called IL2RP)) and the IL-2 receptor gamma subunit (IL2Rg (also called IL2Ry)) A first CISC component and a second CISC component are also described, which contain the functional portion of the domain.

Methods for assessing the phenotype and function of Treg cells, including cells in which FoxP3 overexpression has been induced, are known in the art (e.g., WO/2018/080541; WO/2019/210078; McMurchy et al. 946: 115-132; Thornton et al., 2019 Eur. J. Immunol. 49:398-412; Aarts-Riemens et al., 2008 Eur. J. Immunol. -1390; McGovern et al., 2017 Front. Immunol. 8: Art. 1517; both of which are expressly incorporated herein by reference in their entirety); It is stated. These methods and their associated methodologies can also be used to characterize the airT cells of the invention described herein.

The airT cells disclosed herein differ from natural Treg cells in that the cytosine at specific positions of the cytosine-guanine (CG) dinucleotide of the intra-intronic Treg-specific demethylated region (TSDR) of the FoxP3 locus. (C) Most of the nucleotides are methylated. For example, in the airT cells of the present invention, at least 80%, 85%, 90%, 95%, 96%, 97% of C nucleotides at nucleotide positions containing demethylated C nucleotides in the TSDR region of native Treg cells , 98% or 99% methylated. Analysis of methylation of the TSDR region of the FoxP3 gene is known to be routine in the art, and several methodologies exist (for example, Salazar et al., 2017 Front. Immunol. 8:219; Ngalamika et al., 2014 Immunol. Invest. 44(2): 126-136; these documents are expressly incorporated herein by reference in their entireties).

Despite the difference in epigenetic modification in the TSDR region between the airT cells of the present invention and natural Treg cells, the airT cells of the present invention are stimulated by specific antigen recognition by the TCR. can elicit immunosuppression in response to Thus, co-transfection of CD4+ cells with a viral vector encoding a FoxP3 construct and a viral vector encoding a TCR construct did not result in Treg-like cells exhibiting antigen-specific suppressive function reported by Wright et al. 2009 Proc. Nat. Acad. Sci. USA 106: 19078), it was unexpected that the airT cells disclosed herein exhibit antigen-specific immunosuppressive properties. Thus, without wishing to be bound by any particular theory, the airT cells disclosed herein are based, at least in part, on the methods of making airT cells, including artificial gene editing, described herein. It seems to have unexpected advantages.

T cell receptor (TCR)
The introduced polynucleotide encoding an exogenous TCR expressed in the airT cells of the invention may contain artificial modifications of the native TCR locus (e.g., TRAC) and/or Artificial alterations of chromosomal sites other than loci may also be included. gene by knocking in (inserting) a nucleic acid molecule comprising a polynucleotide encoding an exogenous TCR at a chromosomal site other than the native TCR locus, such as, for example, the FOXP3 locus or AAVS1 locus, or another locus May be edited.

In a specific embodiment, in the airT cells of the invention, two genes each giving rise to a TCR gene product co-expressed with a first CISC component and a FoxP3 gene product co-expressed with a second CISC component Gene editing may be designed to occur at separate alleles of the same locus (e.g., double-editing methods that edit two alleles) or may be designed to occur at two different loci ( For example, the double-editing method, which edits two loci). Exemplary TCR amino acid sequences that specifically recognize antigens associated with the etiology of allergic, autoimmune and/or inflammatory diseases and the nucleotide sequences encoding them are disclosed herein (e.g. , see FIGS. 136-144).

Gene editing As used herein, a "chromosomal gene knockout" prevents (e.g., reduces, delays, represses, inhibits) the production of a functionally active endogenous polypeptide product by a host cell. ), refers to the modification or inactivation of a gene in a host cell or the introduction of an inhibitory factor into the host cell. Genetic modifications that result in knockout or inactivation of chromosomal genes include, for example, nonsense mutations (including formation of premature stop codons), missense mutations, introduction of gene deletions or strand breaks, and introduction of endogenous genes in host cells. Heterologous expression of inhibitory nucleic acid molecules that suppress expression are included.

In certain embodiments, chromosomal gene knockouts or chromosomal gene knockins (eg, insertions) are performed by editing the chromosome of the host cell. Chromosome editing can be performed using, for example, endonucleases. As used herein, "endonuclease" refers to an enzyme that can catalyze the cleavage of phosphodiester bonds within polynucleotide chains. In certain embodiments, the target gene can be inactivated or "knocked out" by cleaving the target gene with an endonuclease. Endonucleases can be natural endonucleases, recombinant endonucleases, genetically engineered endonucleases, fusion endonucleases. Examples of endonucleases used in gene editing include zinc finger nucleases (ZFNs), TALE nucleases (TALENs), CRISPR-Cas nucleases, meganucleases and megaTALs.

Nucleic acid strand breaks caused by endonucleases are usually double-strand breaks (DSBs), which are divided into two groups: homologous recombination repair (HDR) or non-homologous end joining (NHEJ). It is often repaired by different mechanisms (NHEJ: Ghezraoui et al., 2014 Mol Cell 55(6):829-842; HDR: Jasin and Rothstein, 2013 Cold Spring Harb Perspect Biol 5(11):a012740, PMID 24097900 ). In homologous recombination repair (HDR) or homologous gene recombination, a donor nucleic acid molecule may be used to "knock in" a donor gene and a donor nucleic acid molecule may be used to "knock out" a target gene. Alternatively, the target gene may be inactivated by knocking in the donor gene or knocking out the target gene using the donor nucleic acid molecule. Non-homologous end joining (NHEJ) is an error-prone repair method that often involves alterations, eg, substitutions, deletions or additions of at least one nucleotide, at the cleavage site of a DNA sequence. Non-homologous end joining may be used to "knock out" the target gene. Although homologous recombination repair often occurs when the donor template is present when the double-strand break occurs, homologous recombination repair is also preferred as the gene editing mechanism for certain embodiments described herein.

As used herein, a "zinc finger nuclease (ZFN)" refers to a fusion protein in which a zinc finger DNA binding domain is fused to a non-specific DNA cleavage domain (such as Fok I endonuclease). Each zinc finger motif, approximately 30 amino acids long, binds approximately 3 base pairs of DNA, and specific amino acid residues can be altered to alter the specificity of triplet sequences (see, eg, Desjarlais et al., Proc. Natl. Acad. Sci. 90:2256-2260, 1993; Wolfe et al., J. Mol. Biol. 285:1917-1934, 1999). Linking multiple zinc finger motifs allows specific binding to a desired DNA sequence (eg, a region about 9-18 base pairs in length). This technology involves ZFNs participating in genome editing by catalyzing the formation of site-specific DNA double-strand breaks (DSBs) in the genome, which in turn generate flanking sequences homologous to the double-strand break sites in the genome. It is based on the fact that a transgene containing a is targeted to integrate into this cleavage site by homologous recombination repair (HDR). Alternatively, ZFN-formed double-strand breaks can result in target gene knockout via repair by non-homologous end joining (NHEJ), which is error-prone. A cellular repair pathway that results in the insertion or deletion of nucleotides at the cleavage site. In certain embodiments, gene knockout or inactivation comprises insertions, deletions, mutations or combinations thereof introduced using ZFN molecules.

As used herein, "transcription activator-like effector nuclease (TALEN)" refers to a fusion protein comprising a TALE DNA binding domain and a DNA cleavage domain (such as Fok I endonuclease). A "TALE DNA binding domain" or "TALE" comprises one or more TALE repeat domains/units, each TALE repeat domain typically having a highly conserved sequence of 33-35 amino acid residues. , the 12th and 13th amino acids are highly variable. This TALE repeat domain is involved in TALE binding to target DNA sequences. A variable amino acid residue is called RVD (Repeat Variable Diresidue) and is involved in specific nucleotide recognition. The natural (canonical) code for TALE recognition of DNA is known and TALE binds to cytosine (C) when positions 12 and 13 of TALE are HD sequences (histidine-aspartic acid) If the 12th and 13th TALEs are NG (asparagine-glycine), the TALE is bound to T nucleotides, and if the 12th and 13th TALEs are NI (asparagine-isoleucine), the TALE is bound to A nucleotides. If the 12th and 13th positions of the TALE are NN (asparagine-asparagine), the TALE binds to G or A nucleotides, and if the 12th and 13th positions of the TALE are NG (asparagine-glycine), the TALE is T Binds to nucleotides. Non-canonical (atypical) RVDs are also known (see, e.g., U.S. Patent Publication No. 2011/0301073; the atypical RVDs described in this document are incorporated herein by reference in their entirety. cited). TALENs can be used to induce site-specific double-strand breaks (DSBs) in the T-cell genome. Non-homologous end joining (NHEJ) ligates the DNA on either side of the double-strand break, with little or no sequence overlap for annealing, thus knocking out gene expression. An error is introduced that causes Alternatively, homologous recombination repair (HDR) can introduce a transgene at the site of a double-strand break when the donor template containing the transgene is flanked by homologous sequences. In certain embodiments, gene knockouts comprise insertions, deletions, mutations or combinations thereof introduced using TALEN molecules.

As used herein, a "clustered regularly interspaced short palindromic repeats/Cas (CRISPR/Cas)" nuclease system refers to a system that employs a CRISPR RNA (crRNA)-guided Cas nuclease, where the 3' end of a complementary target sequence Shortly after, it uses base-pair complementarity to recognize target sites in the genome (known as protospacers) and cleave DNA if a conserved short protospacer-associated motif (PAM) is present. . CRISPR/Cas systems are classified into three classes (ie, Types I, II and III) based on the sequence of the Cas nuclease and its structure. Type I and III crRNA-guided surveillance complexes require multiple Cas subunits. The most studied type II CRISPR/Cas system contains at least three components: RNA-guided Cas9 nuclease, crRNA and transactivating crRNA (tracrRNA). The tracrRNA contains a double-strand forming region. crRNA and tracrRNA form a duplex that can interact with the Cas9 nuclease, via Watson-Crick base pairing between the spacer on crRNA and the protospacer on the target DNA upstream of PAM. The Cas9/crRNA:tracrRNA complex is directed to specific sites in target DNA. Within the region defined by this crRNA spacer, a double-strand cut is made by the Cas9 nuclease. In repair by non-homologous end joining (NHEJ), insertions and/or deletions at the target locus disrupt expression at the target locus. Alternatively, homologous recombination repair (HDR) can introduce a transgene flanked by homologous sequences in the donor template at the site of a double-strand break. Single-stranded guide RNAs (also called sgRNAs or gRNAs) can be generated from crRNA and tracrRNA (see, eg, Jinek et al., Science 337:816-21, 2012). Additionally, the region of the guide RNA complementary to the target site can be altered or programmed so that the desired sequence can be targeted (Xie et al., PLOS One 9:e100448, 2014; US patent application Publication No. 2014/0068797; U.S. Patent Application Publication No. 2014/0186843; U.S. Patent No. 8,697,359; and PCT Publication No. WO 2015/071474; all of which are incorporated herein by reference).

In certain embodiments, gene knockout or inactivation comprises insertions, deletions, mutations or combinations thereof introduced using the CRISPR/Cas nuclease system. US Patent Publication No. 2016/033377, which is expressly incorporated herein by reference in its entirety, teaches a method for enhancing gene editing using endonucleases, which method comprises , including the use of AAV-expressed guide RNAs in CRISPR/Cas (eg, Cas9) gene editing systems. Exemplary gRNA sequences for knocking out endogenous genes encoding proteins of immune cells and methods using these sequences include Ren et al., Clin. Cancer Res. 23(9):2255-2266 (2017). ), and all of the gRNA, CAS9 DNA, vectors and gene knockout technology described in this document are expressly incorporated herein by reference in their entireties.

As used herein, "meganuclease", also called "homing endonuclease", refers to an endodeoxyribonuclease characterized by a long recognition site (a double-stranded DNA sequence of about 12-40 base pairs). Meganucleases can be classified into five families based on their sequences and structural motifs: LAGLIDADG, GIY-YIG, HNH, His-Cys box and PD-(D/E)XK. Typical meganucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI , I-TevI, I-TevII and I-TevIII, the recognition sequences of which are known (e.g., US Pat. No. 5,420,032; US Pat. No. 6,833,252; Belfort et al., Nucleic Acids Res. 25 :3379-3388, 1997; Dujon et al., Gene 82:115-118, 1989; Perler et al., Nucleic Acids Res. 22:1125-1127, 1994; Jasin, Trends Genet. Gimble et al., J. Mol. Biol. 263:163-180, 1996; Argast et al., J. Mol. Biol. 280:345-353, 1998).

In certain embodiments, targets selected from genes encoding PD-1, LAG3, TIM3, CTLA4, TIGIT, FasL, HLA, and genes encoding components of TCR by using natural meganucleases may prompt site-specific genomic modification of In another embodiment, recombinant meganucleases with novel binding specificities for target genes are used to perform site-specific genomic modifications (e.g., Porteus et al., Nat. Biotechnol. 23:967- 73, 2005; Sussman et al., J. Mol. Biol. 342:31-41, 2004; Epinat et al., Nucleic Acids Res. 31:2952-62, 2003; 895-905, 2002; Ashworth et al., Nature 441:656-659, 2006; Paques et al., Curr. Gene Ther. 7:49-66, 2007; US Patent Publication No. 2007/0117128; US Patent Publication No. 2006/0153826; US Patent Publication No. 2006/0078552; and US Patent Publication No. 2004/0002092). In a further embodiment, a homing endonuclease is added with the DNA binding domain module of TALEN to create a fusion protein known as megaTAL, which is used to knock out chromosomal genes. megaTAL not only knocks out or inactivates one or more target genes, but also introduces (knocks in) heterologous or exogenous polynucleotides in combination with an exogenous donor template encoding a polypeptide of interest. can also

Chromosomal gene knockouts can be confirmed directly by DNA sequencing of host immune cells after performing the knockout procedure or using a knockout agent. Chromosomal gene knockout can also be inferred from the absence of gene expression following knockout (eg, the absence of mRNA or polypeptide product encoded by the knocked-out gene).

In certain embodiments, the knockout or inactivation of a chromosomal gene is the knockout or inactivation of a TCR component gene selected from TCRα variable region genes, TCRβ variable region genes, TCR constant region genes, and combinations thereof. including transformation.

T cells and TCRs
The CD4+CD25+air T cells of the invention comprise an artificially modified FOXP3 gene as described herein, and additionally at least one transduced gene encoding an antigen-specific T cell receptor (TCR) polypeptide. containing polynucleotides. In certain embodiments, the native TCR gene is preferably knocked out, eg, the TCRα (TRAC) locus is preferably knocked out by targeted gene editing. As used herein, "knockout" may refer to inactivation of a gene and/or gene product, e.g., inactivating a gene and/or gene product by deletion of the gene or part thereof; Or it may refer to interfering with the transcription and/or translation of a gene and/or gene product by inserting a nucleic acid into the gene. Furthermore, in certain embodiments, it is preferred that the introduced polynucleotide encoding the TCR has been knocked into a specific locus, such as the TRAC locus or another target locus, by gene editing.

Accordingly, the airT cells of the present invention are, in preferred embodiments, artificial immunoregulatory T cells produced by selectively editing one or more specific loci of T lymphocytes as described herein. include. T cells derived from mammals are preferred, such as humans, non-human primates (e.g., chimpanzees, rhesus monkeys, gorillas, etc.), rodents (e.g., mice, rats, etc.), lagomorphs (e.g., T cells obtained from rabbits, hares, pikas, etc.), ungulates (eg, cows, horses, pigs, sheep, etc.), or other mammals are preferred. In certain preferred embodiments, said T cells are human T cells.

T-cells or T-lymphocytes are immune system cells that mature in the thymus and produce T-cell receptors (TCRs), which, for example, are usually composed of αβ- or γδ-heterodimers. is a cell surface receptor consisting of an antigen-specific heterodimer that A given T cell clone usually expresses only a single clonal TCR that recognizes a specific antigenic epitope presented by syngeneic antigen-presenting cells via major histocompatibility complex-encoded determinants. . T cells are naive T cells (“TN”; T cells that have not been exposed to antigen; (as described herein) expression of CD62L, CCR7, CD28, CD3, CD127 and CD45RA compared to TCM increased, decreased or absent CD45RO expression), memory T cells (TM) (antigen-experienced, long-lived T cells) (including stem cell memory T cells) , effector cells (antigen-experienced, cytotoxic T cells). TMs are composed of central memory T cells (TCM) (T cells expressing CD62L, CCR7, CD28, CD95, CD45RO and CD127) and effector memory T cells (TEM) (expressing CD45RO, CD62L, CCR7, CD28 and CD45RA). T cells with decreased expression) can be further divided into two subsets. Effector T cells (TE) refer to antigen-experienced CD8+ cytotoxic T lymphocytes, express CD45RA, have reduced expression of CD62L, CCR7 and CD28 compared to TCM, and have granzymes and perforin. is positive. Helper T cells (TH) are CD4+ cells that influence the activity of other immune cells by releasing cytokines. CD4+ T cells can activate or suppress adaptive immune responses, and which of these two functions is induced depends on the presence of other cells and other signals. T cells can be collected using known techniques, e.g., using antibodies that specifically recognize one or more T cell surface phenotypic markers, e.g. Various subpopulations or combinations thereof can be enriched or depleted by sexual binding, flow cytometry, fluorescence-activated cell sorting (FACS), or immunomagnetic bead selection. Other typical T cells include regulatory T cells (Treg), such as CD4+CD25+ (Foxp3+) regulatory T cells and Treg17 cells (also known as suppressor T cells), as well as Tr1 cells, Th3 cells, CD8+CD28- cells, and Qa-1 restricted T cells.

As used herein, "T-cell receptor (TCR)" refers to a member of the immunoglobulin superfamily that has a variable binding domain, constant domain, transmembrane region and short cytoplasmic tail. See, eg, Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. TCRs are capable of specifically binding antigenic peptides bound to major histocompatibility complex (MHC)-encoded receptors. TCRs are found on the surface of T cells, but can also be released into the extracellular environment in soluble form and are usually heterodimers with α and β chains (also known as TCRα and TCRβ). or heterodimers with gamma and delta chains (also known as TCRgamma and TCRdelta), where the alpha, beta, gamma, and delta chains are all characteristic of each chain It has a constant (C) region and a highly diverse variable (V) region, and the variable (V) region contains a complementarity determining region ( CDRs) are present. In certain embodiments, a polynucleotide encoding a TCR directs expression in a particular host cell, e.g., immune system cells, hematopoietic stem cells, T cells, primary T cells, T cell lines, NK cells, natural killer T cells. Codons can be optimized to improve (Scholten et al., Clin. Immunol. 119:135, 2006).

Exemplary T cells capable of expressing TCRs encoded by heterologous genetic material introduced into cells according to certain embodiments of the present disclosure include CD4+ T cells, CD8+ T cells and their related subpopulations. (eg, naive T cells, central memory T cells, stem cell memory T cells, effector memory T cells). In a preferred embodiment, the exemplary T cells are CD4+ T cells, and the genetic The compilation introduces genetic material encoding a TCR, which is a TCR that specifically recognizes antigens associated with the pathogenesis of autoimmune, allergic or inflammatory diseases, e.g. or a TCR of the present disclosure that specifically recognizes a T-cell epitope of a particular autoantigen, allergen or inflammatory disease antigen.

The extracellular portion of the TCR chains (e.g., the α and β chains), as well as other antigen-binding members of the immunoglobulin superfamily (e.g., immunoglobulins, also called antibodies), contain N-terminal variable domains (e.g., α-chain variable domain (Vα), β-chain variable domain (Vβ); usually Kabat numbering (Kabat et al., “Sequences of Proteins of Immunological Interest”, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.) and one constant domain adjacent to the cell membrane (e.g., the α-chain constant domain (Cα), usually 117 according to Kabat numbering). It contains two immunoglobulin domains, consisting of amino acids ∼259; the β-chain constant domain (Cβ), usually consisting of amino acids 117-295, based on Kabat numbering. Furthermore, like immunoglobulins, variable domains contain multiple complementarity determining regions (CDRs) separated from one another by framework regions (FRs) (see, for example, Jores et al., Proc. Nat'l Acad. USA 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). . TCRs used in this disclosure may be obtained from various types of animals such as humans, non-human primates, mice, rats, rabbits, and other mammals.

A "variable region" or "variable domain" is a structural domain of an immunoglobulin superfamily binding protein (e.g., the α or β chain (or the γ and δ chains of a γδTCR)). The α- and β-chain variable domains (Vα) and β-chain variable domains (Vβ) of native TCRs generally have similar structures, with each variable domain comprising four generally conserved framework regions ( FR) and three CDRs. The Vα domain is encoded by two separate DNA segments, the variable and joining gene segments (V–J), and the Vβ domain is encoded by three separate DNA segments: the variable, diversity and joining gene segments (V–D–J). encoded by a DNA segment of A single Vα or Vβ domain alone may be sufficient to confer antigen binding specificity. Further, the Vα or Vβ domain of a TCR that binds to a specific antigen is used to screen a library of complementarity determining regions of the Vα or Vβ domain, respectively, to isolate a TCR that binds to said specific antigen. may

The term "complementarity determining region" or "CDR" is used interchangeably with "hypervariable region" or "HVR" and immunoglobulin (e.g. TCR) variable regions that confer antigen specificity and/or binding affinity It is known in the art to refer to an amino acid sequence within a primary amino acid sequence in which multiple CDRs are separated from each other by framework regions. Typically, each TCR α chain variable region has three CDRs (αCDR1, αCDR2, αCDR3) and each TCR β chain variable region has three CDRs (βCDR1, βCDR2, βCDR3). In the TCR, the major CDR responsible for recognition of processed antigen is thought to be CDR3. Usually CDR1 and CDR2 primarily interact with MHC or only CDR1 and CDR2 interact with MHC.

CDR1 and CDR2 are encoded within the variable gene segment of the sequence encoding the variable region of the TCR, CDR3 is the variable segment-to-joining segment (V-J) of Vα, or the variable segment to diversity segment of Vβ. and a region spanning the joining segment (V-D-J). Thus, if Vα variable gene segments or Vβ variable gene segments have been identified, their corresponding CDR1 and CDR2 sequences can be deduced, for example, according to the numbering scheme described herein. CDR3 is typically significantly more diverse than CDR1 and CDR2 due to the addition and/or deletion of nucleotides during the recombination process.

Variable domain sequences of TCRs are aligned by a numbering scheme (e.g., Kabat, Chothia, EU, IMGT, Enhanced Chothia and Aho) using, e.g., the ANARCI software tool (2016, Bioinformatics 15:298-300). , annotation of the same residue positions, and comparison of different molecules can be performed. Utilization of a numbering scheme allows for standardized definition of the framework regions and CDRs of TCR variable domains. In certain embodiments, the CDRs of this disclosure are identified according to the IMGT numbering scheme (Lefranc et al., Dev. Comp. Immunol. 27:55, 2003; imgt.org/IMGTindex/V-QUEST.php). .

As used herein, "CD4" is a glycoprotein that functions as an immunoglobulin co-receptor that assists in the exchange of information between the TCR and antigen-presenting cells (Campbell & Reece, Biology 909 (Benjamin Cummings, Sixth Ed. , 2002)). CD4 is found on the surface of immune cells such as helper T cells, monocytes, macrophages and dendritic cells and contains four immunoglobulin domains (D1-D4), which are expressed on the cell surface. During antigen presentation, CD4 is recruited along with the TCR complex, and CD4 and the TCR complex bind to different regions of the MHC class II molecule (CD4 binds to MHCIIβ2 and the TCR complex binds to MHCIIα1/β1). do). Without wishing to be bound by theory, the location of CD4 adjacent to the TCR complex suggests that CD4-related kinase molecules activate immunoreceptor tyrosine-activating motifs present in the cytoplasmic domain of CD3. (ITAM) phosphorylation is thought to be possible. This activity is thought to enhance the immune response by amplifying the signals generated by activated TCRs to generate or recruit various types of immune cells, including helper T cells. .

In certain embodiments, TCRs are found on the surface of T cells (ie, T lymphocytes) and associate with the CD3 complex. "CD3" is a multi-protein complex composed of six chains (Abbas and Lichtman, 2003; see Janeway et al., p.172 and p.178, 1999) and is an antigen in T cells. Involved in signal transduction. In mammals, the CD3 complex comprises a CD3 gamma chain, a CD3 delta chain, two CD3 epsilon chains, and a homodimer of CD3 zeta chains. The CD3 γ, CD3 β and CD3 ε chains are related to each other as cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane regions of the CD3 γ, CD3 β and CD3 ε chains are negatively charged, which associates them with the positively charged regions of the T cell receptor chain. is thought to be possible. The cytoplasmic tails of the CD3γ, CD3β and CD3ε chains each contain one conserved motif known as the immunoreceptor tyrosine activation motif (ITAM), whereas each CD3ζ chain has three of these motifs. Without wishing to be bound by theory, ITAMs are believed to be important for the signaling ability of the TCR complex. CD3 used in the present disclosure may be obtained from various types of animals such as humans, non-human primates, mice, rats and other mammals.

As used herein, "TCR complex" refers to a complex formed by the association of CD3 and TCR. For example, the TCR complex may consist of a CD3 gamma chain, a CD3 beta chain, two CD3 epsilon chains, a homodimer of CD3 zeta chains, a TCR alpha chain and a TCR beta chain. Alternatively, the TCR complex may consist of a CD3 gamma chain, a CD3 beta chain, two CD3 epsilon chains, a homodimer of CD3 zeta chains, a TCR gamma chain and a TCR beta chain.

As used herein, a "TCR complex component" refers to a TCR chain (i.e. TCRα, TCRβ, TCRγ or TCRδ), a CD3 chain (i.e. CD3γ, CD3δ, CD3ε or CD3ζ), or two or more TCR chains Alternatively, a complex formed by CD3 chains (for example, a complex consisting of TCRα and TCRβ, a complex consisting of TCRγ and TCRδ, a complex consisting of CD3ε and CD3δ, a complex consisting of CD3γ and CD3ε, or TCRα, TCRβ, CD3γ , CD3δ and the TCR partial complex consisting of two CD3ε chains).

As used herein, a "chimeric antigen receptor (CAR)" refers to two or more naturally occurring amino acid sequences, domains or motifs joined in a manner not found in nature or in a host cell Refers to an artificial fusion protein that can function as a receptor when expressed on the surface of a cell, such as a T cell. A CAR is an extracellular portion (e.g., immunoglobulin or extracellular portions obtained from, or derived from, immunoglobulin-like molecules; e.g., TCR antigen-binding domains obtained from TCRs specific for autoantigens, allergens or antigens associated with inflammatory diseases; or TCR antigen-binding domains derived from these; scFv obtained from antibodies or scFv derived from antibodies; antigen-binding domains obtained from killer immune receptors of NK cells or antigen-binding domains derived therefrom, etc.) Sadelain et al., Cancer Discov., 3(4):388 (2013); Harris and Kranz, Trends Pharmacol. Sci., 37(3):220 (2016), Stone et al. al., Cancer Immunol. Immunother., 63(11):1163 (2014) and Walseng et al., Scientific Reports 7:10713 (2017); incorporated herein by reference).

Many of the polypeptides encoded by polynucleotide sequences may contain a "signal peptide" (also known as a leader sequence, leader peptide or transit peptide). The signal peptide directs the newly synthesized polypeptide to the appropriate intracellular or extracellular location. A signal peptide may be removed from a polypeptide during biosynthesis, removed from the polypeptide after positioning the polypeptide in the proper location within the cell, or after the polypeptide has been secreted out of the cell. good too. A polypeptide with a signal peptide is referred to herein as a "pre-protein" and a polypeptide from which the signal peptide has been removed is referred to herein as a "mature" protein or "mature" polypeptide.

A "linker" refers to an amino acid sequence that joins two proteins, polypeptides, peptides, domains, regions or motifs such that the polypeptide resulting from linkage via the linker retains a specific binding affinity for a target molecule. A spacer function may be provided that is compatible with the interaction of the two linked domains so as to (eg scTCR) or retain signaling activity (eg the TCR complex). In certain embodiments, the linker is composed of about 2-35 amino acids or 2-35 amino acids, such as about 4-20 amino acids or 4-20 amino acids, about 8-15 1 amino acid or 8-15 amino acids, or about 15-25 amino acids or 15-25 amino acids. Typical linkers include glycinethrin linkers known in the art.

Exemplary TCR V region sequences of TCRs that specifically recognize antigens associated with the autoimmune, allergic and inflammatory diseases described herein and the polynucleotide sequences encoding same are provided herein. The disclosure also includes what is described in the examples and drawings. Further disclosed herein, including the examples and figures, are polypeptide sequences comprising antigenic epitopes of antigens recognized by the TCR that are associated with the autoimmune, allergic and inflammatory diseases described herein. ing.

"Antigen" generally refers to an immunogenic molecule that provokes an immune response. This immune response includes the production of antibodies that specifically bind to antigens, or the activation of specific immunocompetent cells (e.g., T cells such as helper T cells, effector T cells, Treg cells), or both. You can Antigens are often thought of as "non-self" structures that the host's immune system recognizes as foreign and exhibits responsiveness, but in the present disclosure, "antigens" are not limited to such non-self antigens, In certain embodiments, autoantigens (also referred to as "self" molecules, "self" cells, "self" organs, or "self" tissue structures) to which the host's immune system is inappropriately responsive in autoimmune diseases. include. Antigens (immunogenic molecules) can be, for example, peptides, glycopeptides, polypeptides, glycopolypeptides, polynucleotides, polysaccharides, lipids, and the like. It is readily understood that an antigen can be artificially synthesized, produced by recombinant technology, or obtained from a biological sample. Typical biological samples that may contain one or more antigens include tissue samples, cells, bodily fluids, biopsies, primary cultures, or combinations thereof. Antigens can be produced by cells that have been modified or genetically engineered to express the antigen, or immunogenic mutations or polymorphisms (e.g., without human intervention or modification) can be produced by cells that endogenously express the

In certain embodiments, the T cells provided herein encode one or more heterologous polynucleotides, including regulatory sequences (e.g., promoters, enhancers, etc.), desired TCRs, as described herein. and/or host cells that may be modified to contain nucleic acid sequences encoding all or part of the FoxP3 transcription factor. Methods for transfecting/transducing T cells with desired nucleic acids have been previously reported (e.g., US Patent Application Publication No. 2004/0087025) and adoptive T cells with desired target specificity are used. It has also been reported that transfer can be performed (e.g. Schmitt et al., Hum. Gen. 20:1240, 2009; Dossett et al., Mol. Ther. 17:742, 2009; Till et al., Blood 112:2261, 2008; Wang et al., Hum. Gene Ther. 18:712, 2007; Kuball et al., Blood 109:2331, 2007; U.S. Patent Publication No. 2011/0243972; Leen et al., Ann. Rev. Immunol. 25:243, 2007), and it is also envisioned, based on the teachings herein, that such methodologies be employed in the embodiments disclosed herein. there is A particularly preferred embodiment relates to artificially modifying the T cell genome by targeted gene editing as described herein.

Transfection or transduction of cells, eg, T cells, or administration of polynucleotides or compositions according to the methods of the invention may be performed using any suitable method. Known methods of delivering polynucleotides to host cells include, for example, the use of cationic polymers, lipid-like molecules or certain commercial products such as IN-VIVO-JET PEI. Other methods include ex vivo transduction, injection, electroporation, DEAE-dextran, ultrasound delivery, liposome-mediated transfection, receptor-mediated transduction, microprojectile bombardment, and transposon-mediated transfer. are mentioned. Methods of transfecting or transducing host cells further include using vectors described herein and known in the art.

Nucleic acids may include DNA or RNA, and may be wholly or partially synthetic. Reference to the nucleotide sequences described herein includes DNA molecules having the sequences described herein, and having the sequences described herein with U substituted for T, unless otherwise stated. Also includes RNA molecules. As used herein, "isolated polynucleotide" means genomic polynucleotides, cDNA polynucleotides, synthetic polynucleotides, combinations thereof, and isolated polynucleotides, depending on their origin ( (2) linked to a polynucleotide with which it is not linked in nature; or (3) ) exist as part of long sequences not found in nature.

"Operatively linked" means that the components to which the term applies are in a relationship such that, under appropriate conditions, they are capable of performing their intrinsic function. For example, a transcriptional regulatory sequence "operably linked" to a coding sequence for a protein is added to the protein-coding sequence such that the protein-coding sequence is expressed under conditions compatible with the transcriptional activity of the transcriptional regulatory sequence. ligated.

As used herein, "regulatory sequence" refers to a polynucleotide sequence that, when ligated or operably linked to a coding sequence, can affect the expression, processing or subcellular localization of the coding sequence. . The properties of such regulatory sequences may depend on the host organism. In certain embodiments, prokaryotic transcription regulatory sequences include promoters, ribosome binding sites and transcription termination sequences. In another specific embodiment, eukaryotic transcription regulatory sequences include promoters that contain one or more sites that recognize transcription factors, transcription enhancer sequences, transcription termination sequences or polyadenylation sequences. In certain embodiments, "regulatory sequences" may include leader sequences and/or fusion partner sequences.

In certain embodiments, the airT cells of the invention may be genetically edited to express the FoxP3 gene product encoded by a FoxP3-encoding nucleotide sequence operably linked to a constitutive promoter. . Here, "constitutive expression of the FoxP3 gene product" refers to an expression level of FOXP3 equal to or higher than that of natural regulatory T (Treg) cells. In certain preferred embodiments, said constitutive promoter is the MND promoter, and in certain preferred embodiments, the MND promoter has been knocked into the native FOXP3 locus by gene editing via homologous recombination repair. In a specific embodiment, said constitutively active promoter is knocked into the native FOXP3 locus downstream of an intronic regulatory T cell (Treg)-specific demethylation region (TSDR). In certain embodiments, a nucleic acid molecule comprising a polynucleotide encoding exogenous FOXP3 and a constitutive promoter operably linked thereto is knocked into the native FOXP3 locus by gene editing via homologous recombination repair. It is In certain embodiments, a nucleic acid molecule comprising a polynucleotide encoding an exogenous FOXP3 and a constitutive promoter operably linked thereto is transformed into a gene other than the native FOXP3 locus by gene editing via homologous recombination repair. Knocked in at a chromosomal site (TRAC locus, AAVS1 locus, another locus, etc.). Accordingly, the present disclosure provides, in these and related embodiments, a gene expression such that the expression of the FOXP3 gene is regulated by a constitutively active promoter (in a particularly preferred embodiment, the constitutively active MND promoter). It is the first teaching that the artificial editing of .RTM.

The term "polynucleotide" as referred to herein means a single- or double-stranded nucleic acid polymer. In certain embodiments, the nucleotides contained in the polynucleotide may be ribonucleotides, deoxyribonucleotides, or modified forms of ribonucleotides or deoxyribonucleotides. Modifications of ribonucleotides or deoxyribonucleotides include base modifications such as bromouridine; ribose modifications such as arabinoside and 2′,3′-dideoxyribose; Modifications of internucleotide linkages such as anilothioates, phosphoraniridates, phosphoramidates and the like can be mentioned. More specifically, "polynucleotide" includes DNA in either single- or double-stranded form.

"Naturally occurring nucleotides" include deoxyribonucleotides and ribonucleotides. "Modified nucleotides" include nucleotides with modified or substituted sugar groups and the like. "Oligonucleotide linkage" includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, etc. be For example, LaPlanche et al., 1986, Nucl. Acids Res., 14:9081; Stec et al., 1984, J. Am. Chem. Soc., 106:6077; Stein et al., 1988, Nucl. ., 16:3209; Zon et al., 1991, Anti-Cancer Drug Design, 6:539; Zon et al., 1991, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, pp. 87-108 (F. Eckstein, Ed. ), Oxford University Press, Oxford England; Stec et al., U.S. Pat. No. 5,151,510; Uhlmann and Peyman, 1990, Chemical Reviews, 90:543. is expressly incorporated herein in its entirety by). Oligonucleotides may include a detectable label that allows detection of the oligonucleotide or its hybridization.

The term "vector" is used to refer to any molecule (eg, nucleic acid, plasmid or virus) that is used to transfer coding information to a host cell. An "expression vector" refers to a vector suitable for transformation of a host cell and contains nucleic acid sequences that direct the expression of and/or regulate the expression of an inserted heterologous nucleic acid sequence. Expression includes, but is not limited to, processes such as transcription, translation and RNA splicing (if introns are present).

As will be readily understood by those of skill in the art, polynucleotides include genomic sequences, plasmid-encoded extragenomic sequences that express or are modified to express proteins, polypeptides, peptides, etc. , and small artificial gene segments may be included. Such segments may be isolated from nature or synthetically modified by one skilled in the art.

Furthermore, as will be readily appreciated by those of skill in the art, a polynucleotide may be single-stranded (coding or antisense strand) or double-stranded, and may be a DNA molecule (genomic DNA, cDNA or synthetic DNA). Or it may be an RNA molecule. RNA molecules may include HnRNA molecules (which contain introns and correspond one-to-one with DNA molecules) and mRNA molecules (which do not contain introns). Additional coding or non-coding sequences may be present within a polynucleotide according to this disclosure, but not necessarily within a polynucleotide according to this disclosure, and a polynucleotide may be another molecule and/or may be bound to a carrier material, but not necessarily to another molecule or carrier material. Polynucleotides may include naturally occurring sequences or may include sequences that encode variants or derivatives of naturally occurring sequences.

In another related embodiment, a polynucleotide variant may have substantial identity with a polynucleotide sequence encoding an immunomodulatory polypeptide described herein. For example, a polynucleotide is a reference polynucleotide sequence (described herein) when measured using a method described herein (e.g., BLAST analysis described below using standard parameters). at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, compared to sequences encoding antibodies) Polynucleotides having the above sequence identities, or sequence identities within the range of any two of these ratios may be used. One skilled in the art can adjust each parameter appropriately, taking into account codon degeneracy, amino acid similarity, reading frame position, etc., to determine the identity of the proteins encoded by the two nucleotide sequences. You can easily understand what you can do.

Polynucleotide variants typically contain one or more substitutions, additions, deletions and/or insertions, but are capable of specifically binding and interacting with another molecule encoded by the polynucleotide variant. , a variant of a polypeptide derived from a given polypeptide, even if it contains one or more such substitutions, additions, deletions and/or insertions of the polynucleotides specified herein. Preferably, its binding affinity is not substantially reduced compared to the polypeptide encoded by the sequence.

In another specific related embodiment, a fragment of a polynucleotide may comprise contiguous sequences of varying lengths identical to or complementary to the sequences encoding the polypeptides described herein, It may consist essentially of such sequences. For example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 contained in a sequence encoding a polypeptide or variant thereof disclosed herein 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75 , 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 300, 400, 500, 1000 or more or at least about 5, about 6, about 7, about 8, about 9 contained in a sequence encoding a polypeptide or variant thereof disclosed herein, About 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22 about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, About 35, about 36, about 37, about 38, about 39, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75 about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 200, about 300, A polynucleotide comprising about 400, about 500, about 1000 or more contiguous nucleotides, or an intermediate length of contiguous nucleotides, or substantially such number of A polynucleotide consisting of contiguous nucleotides is provided. As used herein, "intermediate length" means a length between the numerical values set forth herein, such as 50, 51, 52, 53; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; any integer within the range of 200-500; 500-1,000, etc.; The polynucleotide sequences described herein may be extended by adding nucleotides to one or both ends that are not found in the native sequence. The additional sequences may be 1, 2, 3, 4, 5, 6, 7, 8, 1, 2, 3, 4, 5, 6, 7, 8 at one or both ends of the polynucleotide encoding the polypeptides described herein. It may consist of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides.

In another embodiment, it hybridizes under moderately to highly stringent conditions to a polynucleotide sequence encoding a polypeptide provided herein or a variant thereof, a fragment thereof, or a complementary sequence thereof Polynucleotides are provided that are capable of Hybridization techniques are well known in the field of molecular biology. By way of example, moderately stringent conditions suitable for testing hybridization of a polynucleotide provided herein to another polynucleotide include 5×SSC, 0.5% SDS and 1.0 mM EDTA ( pH 8.0); hybridizing overnight in 5x SSC at 50-60°C; using 2x SSC, 0.5x SSC and 0.2x SSC containing 0.1% SDS, respectively includes two washes for 20 minutes at 65°C. Those skilled in the art will appreciate that stringent hybridization conditions can be readily altered, such as by altering the salt content of the hybridization solution and/or the temperature at which the hybridization is conducted. For example, in another embodiment, suitable highly stringent hybridization conditions are the same as those described above, except that the hybridization temperature is increased to, for example, 60-65°C or 65-70°C. is.

The polynucleotides or fragments thereof described herein may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, or other coding segments, regardless of the length of the coding sequence itself. and its overall length may vary considerably. Thus, it is envisioned that nucleic acid fragments of nearly any length can be used, the total length being preferably limited by the ease of preparation and the recombinant DNA protocol used. For example, as an example, the total length is 10,000 base pairs, 5000 base pairs, 3000 base pairs, 2,000 base pairs, 1,000 base pairs, 500 base pairs, 200 base pairs, 100 base pairs, 50 base pairs, etc., or about 10,000 base pairs. about 5000 base pairs, about 3000 base pairs, about 2,000 base pairs, about 1,000 base pairs, about 500 base pairs, about 200 base pairs, about 100 base pairs, about 50 base pairs, etc. (including all intermediate lengths) ) are expected to be useful.

In a comparison of polynucleotide sequences, two sequences are "identical" if the nucleotide sequences of the two sequences are the same when the two sequences are aligned for maximum correspondence, as described below. ”. Comparisons between two sequences are typically performed by comparing the sequences within a comparison window to identify local regions of sequence homology and comparing them. As used herein, a "comparison window" is defined as at least 20 consecutive or at least 20 consecutive positions for comparing the same number of consecutive positions after optimizing the alignment of a reference sequence and a particular sequence. Refers to a segment consisting of about 20 (usually 30-75 or 40-50) positions.

Optimal alignment of sequences for comparison may be performed using the Megalign program (DNASTAR Inc., Madison, Wis.) included in the Lasergene suite of bioinformatics software with default parameters. This program has been used for several alignment schemes described in various publications, including Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. , Suppl. 3, pp. 345-358, Dayhoff, M.O. (1978) A model of evolutionary change in proteins - Matrices for detecting distant relationships.; Hein J., Unified Approach to Alignment and Phylogenes, pp. 626-645. (1990); Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M., CABIOS 5:151-153 (1989); Myers, E.W. and Muller W., CABIOS 4:11-17 (1988); Robinson, E.D., Comb. Theor 11:105 (1971); Santou, N. Nes, M., Mol. P.H.A. and Sokal, R.R., Numerical Taxonomy - the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA (1973); Wilbur, W.J. and Lipman, D.J., Proc. Natl. Acad., Sci. USA 80:726- 730 (1983).

Alternatively, optimal alignment of sequences for comparison can be performed using the local identity algorithm described in Smith and Waterman, Add. APL. Math 2:482 (1981), Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method described in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 (1988), and computer implementations of these algorithms. (GAP, BESTFIT, BLAST, FASTA and TFASTA in the Wisconsin Genetics Software Package from the Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.) or by inspection.

An example of a preferred algorithm suitable for determining sequence identity and sequence homology is the BLAST algorithm described in Altschul et al., Nucl. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. and the BLAST 2.0 algorithm described in Biol. 215:403-410 (1990). For example, BLAST or BLAST 2.0, using the parameters described herein, can be used to determine sequence identity between two or more polynucleotides. Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information. As an illustrative example, by using M (reward score for a pair of matched residues; always >0) and N (penalty score for mismatched residues; always <0) as parameters for nucleotide sequences, A cumulative score can be calculated. Extension of hit words in both directions is such that when the cumulative alignment score begins to fall from the maximum value by the value of X, the alignment of one or more residues with negative scores accumulates to a cumulative score of 0 or less. or when the end of one sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for comparing nucleotide sequences) has as default parameters wordlength (W) = 11, expectation (E) = 10, the BLOSUM62 scoring matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)), (B) = 50, expectation (E) = 10, M = 5, N = -4, and a comparison of both strands is used.

In certain embodiments, "sequence identity (%)" is determined by comparing two sequences in an optimized alignment within a comparison window comprising at least 20 positions, and the polynucleotide within the comparison window A portion of the sequence is 20% or less (usually 5-15% or 10-12%) compared to the reference sequence (not including additions or deletions) of the optimized alignment of these two sequences. It may contain additions or deletions (ie gaps). Sequence identity (%) is determined by determining the number of positions where identical nucleobases occur in both sequences, calculating the number of positions where the nucleobases match, and dividing the number of matched positions by the number of positions in the reference sequence. % sequence identity is determined by dividing by the total number (ie, the size of the comparison window) and multiplying the result by 100.

As will be appreciated by those of skill in the art, the genetic code is degenerate; There is a wide variety of nucleotide sequences that encode the antibodies described herein that specifically bind. Some of these polynucleotides have minimal sequence identity to the native or original polynucleotide sequences encoding the FoxP3 polypeptides, TCR polypeptides or antigenic polypeptides described herein. do not have. However, polynucleotides that differ due to differences in codon usage are expressly contemplated in this disclosure. Certain embodiments specifically envision sequences that are codon-optimized for expression in mammals.

Thus, in another embodiment of the invention, mutagenesis methods such as site-directed mutagenesis are used in the production of variants and/or derivatives of the FoxP3 polypeptides, TCR polypeptides or antigenic polypeptides described herein. may be used. Mutagenesis methods can introduce specific modifications to polypeptide sequences by introducing mutations into the original polynucleotides encoding these polypeptides. Such techniques are direct methods for generating and testing sequence variants that take into account one or more of the foregoing, for example, by introducing one or more nucleotide sequence changes into a polynucleotide. It provides an effective method.

Site-directed mutagenesis uses a specific oligonucleotide sequence encoding the DNA sequence of the desired mutation, flanked by a sufficient number of nucleotides, to generate a primer sequence of sufficient size and sequence complexity. Mutants can be generated by providing and extending primers from both ends of the deletion junction to form stable duplexes. Mutations may be introduced into a selected polynucleotide sequence to improve, alter, reduce, modify or otherwise alter the properties of the polynucleotide itself and/or the polypeptide encoded by the polynucleotide. may be altered in properties, activity, composition, stability or primary sequence.

In certain embodiments, the inventors have discovered that by introducing mutations into the polynucleotide sequences encoding the FoxP3 polypeptides, TCR polypeptides or antigenic polypeptides disclosed herein or variants thereof, this Contemplates modifying one or more properties of a polypeptide encoded by a polynucleotide sequence, and (e.g., in the case of a TCR or antigenic peptide) directed to a ligand recognized by said polypeptide or variant thereof. It is envisaged to alter the binding affinity of the peptide or its variants, or (eg in the case of FoxP3) immunosuppressive effect. Site-directed mutagenesis techniques are well known in the art and are widely used for the generation of polypeptide and polynucleotide variants. For example, site-directed mutagenesis is often used to modify specific portions of DNA molecules. In such embodiments, primers, such as 14-25 nucleotides in length or about 14-25 nucleotides in length, are typically used to cover about 5-10 residues or 5-10 residues on either side of the junction of the sequences. residues are modified.

As will be appreciated by those skilled in the art, site-directed mutagenesis techniques often employ phage vectors in either single- or double-stranded form. Standard vectors useful for site-directed mutagenesis include vectors such as the M13 phage. These phage are readily available commercially and their use is generally well known to those skilled in the art. Double-stranded plasmids are also routinely used in site-directed mutagenesis that eliminates the step of transferring the gene of interest from the plasmid to the phage.

Site-directed mutagenesis, as described herein, generally involves first obtaining a single-stranded vector that contains within its sequence a DNA sequence encoding the desired peptide, or a DNA sequence that encodes the desired peptide. This is done by melting the double-stranded vector it contains and separating it into two strands. An oligonucleotide primer bearing the desired mutated sequence is prepared, usually synthetically. This primer is then annealed to the single-stranded vector and DNA polymerized by an enzyme such as the Klenow fragment of E. coli polymerase I to synthesize the mutated strand. In this way, a heteroduplex is formed in which the first strand encodes the unmutated original sequence and the second strand carries the desired mutation. This heteroduplex vector is then used to transform suitable cells (E. coli cells) and clones containing the recombinant vector with the mutated sequence are selected.

The preparation of sequence variants of selected peptide-encoding DNA segments using site-directed mutagenesis provides, but is not limited to, the production of potentially useful peptide species. , there are other methods by which it is possible to obtain sequence variants of peptides and the DNA sequences that encode them. For example, sequence variants may be obtained by treating or contacting a recombinant vector encoding the desired peptide sequence with a mutagenic agent such as hydroxylamine. Specific details regarding these methods and protocols are taught in Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby, 1994 and Maniatis et al. incorporated herein by reference for the purpose).

As used herein, "oligonucleotide-directed mutagenesis" refers to template-dependent mutagenesis in which the concentration of a particular nucleic acid molecule is increased over the initial concentration or the concentration of detectable signal (e.g., amplification) is increased. Refers to methods and vector-mediated propagation. As used herein, "oligonucleotide-directed mutagenesis" refers to methods involving template-dependent extension of a molecule of a primer. "Template-dependent methods" are characterized in that the sequence of newly synthesized nucleic acid strands is determined by the well-known rules of complementary base pairing (see, e.g., Watson (1987)). refers to nucleic acid synthesis of RNA or DNA molecules. In general, vector-based methodologies also include introducing nucleic acid fragments into DNA or RNA vectors, amplifying clones of the resulting vectors, and recovering the amplified nucleic acid fragments. An example of such methodology is described in US Pat. No. 4,237,224, which is expressly incorporated herein by reference in its entirety.

Another method of producing variants of a polypeptide may utilize recursive sequence recombination as described in U.S. Pat. No. 5,837,458, which is expressly incorporated herein by reference in its entirety. ). In this method, iterative recombination and screening or selection can be used to "evolve" individual polynucleotide variants with improved binding affinity, for example. In certain embodiments, further provided are constructs in the form of plasmids, vectors, transcription or expression cassettes comprising at least one polynucleotide as described herein.

In practicing some embodiments of the present invention, conventional methods in virology, immunology, microbiology, molecular biology and recombinant DNA technology within the skill of the art are employed unless otherwise indicated. The uses are readily understood, and most of these methods are described below for purposes of illustration. Such techniques are detailed in the literature. Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, N.Y. (2009); Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook and Russell , Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); See Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984), both of which are hereby incorporated by reference in their entireties.

DNA recombination, oligonucleotide synthesis, tissue culture and transformation (eg, electroporation, lipofection, etc.) may be performed using standard techniques. Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications, by methods commonly known in the art, or as described herein. These and related techniques and operations may generally be performed according to conventional methods well known in the art and may be found in various general references or references cited or discussed herein. It may be performed according to the description of the reference literature which is more detailed. Unless specific definitions are given, the nomenclature, tests and techniques used in connection with molecular biology, analytical chemistry, synthetic organic chemistry and medicinal chemistry described herein are those within the skill of the art. It is a known one that is generally used. Also, standard techniques may be used for recombinant techniques, molecular biological synthesis, microbiological synthesis, chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients. good.

Autoimmune, Allergic and Inflammatory Diseases and Antigens Associated Therewith As described above, the airT cells of the present invention can be used to treat and/or alleviate certain autoimmune, allergic and inflammatory diseases. good too. Clinical signs and symptoms and diagnostic criteria for such diseases are known in the art. Human patients or other mammalian hosts in need of antigen-specific immunosuppression may benefit from administration of the airT cells of the present invention for the aforementioned diseases, i.e., clinically inappropriate pro-inflammatory mediators (e.g., , cytokines, lymphokines, hormones, etc.) and/or local or systemically increased inflammatory cells may be found, examples of diseases that individuals have include type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, muscle gravis. Asthenia, rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, bullous pemphigoid, pemphigus vulgaris, autoimmune hepatitis, asthma, allergies (e.g. food allergens, plant allergens, animal allergens, environmental allergens, drug allergens) , specific hypersensitivity to chemical allergens or other allergens), transplant tolerance induction (e.g., islet cell transplantation), and graft-versus-host disease (GVHD) after stem cell (e.g., hematopoietic stem cell) transplantation. but not limited to these.

Some of the antigens associated with these diseases, particularly those for which there are epitopes recognized by the TCR, are known and illustrated in the figures. Also shown in these figures are TCR V region sequences of TCRs shown based on their ability to recognize antigens of the disclosure associated with autoimmune, allergic or inflammatory diseases.

Methods for identifying and characterizing antigens associated with the pathogenesis of autoimmune, allergic or inflammatory diseases, including determination of autoreactive T cell epitopes, are known in the art. For example, exemplary methods for identifying islet autoantigenic polypeptides, including fragments of islet autoantigenic polypeptides recognized by T cells, from subjects with type 1 diabetes (T1D) are described by Cerosaletti et al. (2017). J. Immunol. 199:323; which is expressly incorporated herein by reference in its entirety). Other polypeptide antigens (including determined autoreactive T-cell epitopes) associated with the etiology of autoimmune, allergic or inflammatory diseases are disclosed herein and illustrated in the Figures. include.

Cerosaletti et al. (2017) also describe a typical methodology for determining the structure of T-cell receptors (TCRs) that recognize antigens associated with the pathogenesis of autoimmune, allergic or inflammatory diseases. It is not limited to this methodology. Different antigen-specific structural features of the TCR associated with the pathogenesis of autoimmune, allergic or inflammatory diseases, such as the variable region of the TCR α chain (Vα) and/or the variable region of the TCR β chain ( Vβ), in whole or in part, as well as the polynucleotide sequences encoding them, etc., are disclosed herein, including those depicted in the figures.

Compositions and Methods of Use Accordingly, in certain embodiments disclosed herein, administration of the airT cells described herein as adoptive transfer immunotherapeutic cells results in excessive and/or clinically detrimental It is envisioned to provide antigen-specific immunosuppression to diseases in which antigen-specific immune activity exists. For example, one example, according to certain embodiments, includes adoptively transferring airT cells disclosed herein into a subject (e.g., a patient with an autoimmune disease, allergic disease, or other inflammatory disease). Immunotherapy protocols are envisioned, but not limited to. Adoptive transfer protocols using unselected T cells or selected T cells are known in the art (e.g. Schmitt et al., 2009 Hum. Gen. 20:1240; Dossett et al., 2009 Mol. Ther. 17:742; Till et al., 2008 Blood 112:2261; Wang et al., 2007 Hum. Gene Ther. US2011/0189141; Leen et al., 2007 Ann. Rev. Immunol. 25:243; US2011/0052530, US2010/0310534; Ho et al. Ho et al., 2003 Canc. Cell 3:431), with modifications of these adoptive transfer protocols according to the teachings herein. , may be used to transfer cell populations containing the desired airT cells produced as described herein.

The airT cells of the present invention may be administered using any administration method that is generally accepted as an administration method for drugs having similar utility. The airT cells of the invention are mixed with suitable physiologically acceptable carriers, diluents or additives, such as e.g. aqueous liquids which may contain suitable salts, buffers and/or stabilizers. It can be prepared in the form of a pharmaceutical composition by Administration of airT cells may be by a variety of routes, including intravenous, intrahepatic, intraperitoneal, intragastric, intraarticular, intrathecal, and other routes, and in one preferred embodiment, , administered by intravenous infusion.

The preferred method of administration depends on the nature of the disease to be treated or prevented, and in certain embodiments, the extent, severity, likelihood of onset and/or duration of disease by the specific methods provided herein. may be reduced (eg, statistically significantly reduced compared to an appropriate control condition such as an untreated control), adverse or clinically undesirable disease. Reduction, suppression or delay of such diseases, e.g., reduction, suppression or delay of local or systemic autoimmune activity, allergic activity or other detrimental inflammatory activity is detected after administration of the airT cells of the invention. It is considered effective if Those skilled in the art relevant to the present invention will appreciate the various diagnostic criteria, surgical indication criteria and other criteria applicable to assessing the efficacy of adoptive transfer administration of the immunoregulatory airT cell compositions described herein. Be familiar with clinical criteria. Humar et al., Atlas of Organ Transplantation, 2006, Springer; Kuo et al., Comprehensive Atlas of Transplantation, 2004 Lippincott, Williams &Wilkins; Gruessner et al., Living Donor Organ Transplantation, 2007 McGraw-Hill Professional; et al., Manual of Stem Cell and Bone Marrow Transplantation, 2009 Cambridge University Press; Wingard et al. (Ed.), Hematopoietic Stem Cell Transplantation: A Handbook for Clinicians, 2009 American Association of Blood Banks.

Thus, in some embodiments, the airT cells of the invention are antigen-specific T cells comprising an antigen-specific TCR polypeptide encoded by at least one introduced polynucleotide encoding an antigen-specific TCR polypeptide. A receptor (TCR) may be expressed and can induce antigen-specific immunosuppression in response to HLA-restricted stimulation by an antigen specifically recognized by the TCR polypeptide. Determination of the presence or absence of immunosuppression may be made according to various criteria familiar to those skilled in the art. See, eg, Sakaguchi et al., 2020 Ann. Rev. Immunol. 38:541, which is expressly incorporated herein by reference in its entirety.

For example, multiple mechanisms have been reported that contribute to the suppressive phenotype of Treg cells, including, for example, the CTLA-4 immune checkpoint; expression of immunosuppressive cytokines such as IL-10 and TGF-β; Cytotoxicity to target cells via the /granzyme pathway; induction of indoleamine-2,3-dioxygenase (IDO) and catabolism of tryptophan in target cells; consumption of adenosine by expression of CD73; competition with effector T (T _eff ) cells for IL-2, based on constitutively expressing CD25 (a subunit of the high-affinity receptor of cytoplasm). For example, Verbsky, JW, and Chatila, TA (2014). Chapter 23 - Immune Dysregulation Leading to Chronic Autoimmunity.; Campbell et al. 2020 Cell Metab. 31(1):18-25; Dominguez-Villar et al., 2018 Nat. Immunol. :775-787.

Thus, in some embodiments, antigen-specifically induced immunosuppression is
(i) suppression of one or both activation and proliferation of effector T cells that recognize an antigen specifically recognized by an airT cell TCR comprising said TCR polypeptide encoded by said at least one introduced polynucleotide; ,
(ii) expression of an inflammatory cytokine or inflammatory mediator by effector T cells that recognize an antigen specifically recognized by an airT cell TCR comprising said TCR polypeptide encoded by said at least one introduced polynucleotide; Suppression,
(iii) production of one or more immunosuppressive cytokines or anti-inflammatory products by airT cells, e.g. release of immunosuppressive cytokines or perforin/granzymes, induction of indoleamine-2,3-dioxygenase (IDO); construction of one or more inhibitory mechanisms, such as competition for IL2 or adenosine, tryptophan catabolism, expression of inhibitory receptors by airT cells, and (iv) said TCR polynucleotide encoded by said at least one introduced polynucleotide. One or more of suppression of activation and/or proliferation of effector T cells that do not recognize the antigen specifically recognized by the airT cell TCR containing the peptide may be included.

In certain instances, adoptive transfer immunotherapy using airT cells of the invention (optionally co-administered with at least one additional therapeutic agent) is administered for 1 to 14 days during a 30-day treatment period. may In certain instances, adoptive transfer immunotherapy using airT cells of the invention (optionally co-administered with at least one additional therapeutic agent) is administered for 1 day, 2 days, It may be administered for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days. Different protocols may be appropriate for individual subjects. When administered as described above, an appropriate dosage is an amount of the compound that is capable of detecting a change or alleviation of symptoms, or at least an indication of autoimmune, allergic or other inflammatory immune activity. An amount of a compound that causes a statistically significant reduction of one by at least 10-50% compared to a basal state (e.g., untreated state), wherein said change or alleviation of symptoms or said indication is a specific blood constituent. can be monitored by measuring the amount, e.g., detectable numbers of immune cells and/or other inflammatory cells in the blood, and/or detectable soluble inflammatory mediators, such as proinflammatory cytokines can be monitored by measuring the amount of

Appropriate dosages and treatment regimens can generally provide sufficient amounts of the airT cells of the invention to provide therapeutic and/or prophylactic benefit. The response to such administration is an improved clinical outcome in treated subjects compared to untreated subjects (e.g., more frequent remissions, complete or partial remissions, or longer disease-free survival). A reduction in a pre-existing immune response (e.g., a statistically significant reduction when compared to relevant controls) to an antigen associated with an autoimmune disease, allergic disease or other inflammatory disease described herein is Usually correlates with improved clinical outcome. Such immune responses may generally be assessed by analyzing standard leukocyte and/or lymphocyte surface markers, cytokine expression, cell proliferation, cytotoxicity, or released cytokines, Such analyzes are routine in the art and may be performed using samples taken from the subject before and after treatment.

For example, the dose of the airT cells of the present invention is an amount sufficient to reduce symptoms of an autoimmune disease with clinical significance (e.g., an amount sufficient to significantly reduce symptoms of an autoimmune disease clinically). preferably sufficient to produce a statistically significant and detectable reduction in the symptoms of an autoimmune disease compared to a suitable control disease). The autoimmune diseases include type 1 diabetes, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis, inflammatory bowel disease (IBD), psoriatic arthritis, Crohn's disease, ulcerative colitis, serum Examples include, but are not limited to, negative spondyloarthropathy, Behcet's disease, vasculitis and other autoimmune diseases.

Thus, in some embodiments, airT cells expressing a TCR that specifically recognizes an antigenic epitope associated with an autoantigen associated with type 1 diabetes (T1D) are adoptively transferred into a patient with type 1 diabetes, followed by type 1 A reduction in one or more relevant clinical criteria known in the art used to assess diabetes may be identified. Typical antigens (usually autoantigens) associated with type 1 diabetes and the structures of TCRs that specifically recognize such antigens are described herein.

Common criteria for stage 2 type 1 diabetes include the detection of two or more islet-specific autoantibodies in the patient and an oral glucose tolerance test demonstrating the presence of abnormal glucose metabolism. It may be In some embodiments, the dysglycemia is a fasting blood glucose level of 110-125 mg/dL (6.1-6.9 mmol/L) and a 2-hour postprandial plasma glucose level of 140 mg/dL (7.8 mmol/L) and less than 200 mg/dL (11.1 mmol/L), or a 30-, 60-, or 90-minute postprandial blood glucose level greater than 200 mg/dL during that time. In some embodiments, clinical type 1 diabetes is the presence of symptoms of diabetes (e.g., not known to be at risk of having type 1 diabetes or not having type 1 diabetes). increased dry mouth, frequent urination, and/or unexplained weight loss compared with normal subjects with known defined as a fasting blood glucose level ≥126 mg/dL, an oral glucose tolerance test (OGTT) 2-hour value ≥200 mg/dL, or a hemoglobin A1c level ≥6.5%. (see, eg, Khokhar et al., 2017 Clin. Diabetes 35(3):133.).

Decreased symptoms of rheumatoid arthritis include, for example, malaise; anorexia; low-grade fever; swollen lymph nodes; weakness; swollen joints; Gripping joints, brittle or stiff joints; bilateral joint pain (knuckles (excluding fingertips), wrists, elbows, shoulders, hips, knees, ankles, toes, jaws) pleurisy; burning eyes; itchy eyes; eye discharge; subcutaneous nodules; One or more declines may be determined, but are not limited to these. Diagnostic criteria and clinical monitoring of rheumatoid arthritis patients are well known to those skilled in the art. See, eg, Hochberg et al., Rheumatology, 2010 Mosby; Firestein et al., Textbook of Rheumatology, 2008 Saunders. Diagnostic criteria and clinical monitoring of patients with rheumatoid arthritis and/or other autoimmune diseases are also well known to those skilled in the art. See, for example, Petrov, Autoimmune Disorders: Symptoms, Diagnosis and Treatment, 2011 Nova Biomedical Books; Mackay et al. (Eds.), The Autoimmune Diseases-Fourth Edition, 2006 Academic Press; Brenner (Ed.), Autoimmune Diseases: Symptoms, Diagnosis and Treatment, 2011 Nova Science Pub. Inc.

Recombinant DNA, peptide and oligonucleotide synthesis, immunoassays, tissue culture and transformation (eg, electroporation, lipofection, etc.) may be performed using standard techniques. Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications, by methods commonly known in the art, or as described herein. These and related techniques and manipulations may generally be performed according to conventional methods well known in the art and may include the microbiological techniques, molecular biology techniques cited or discussed herein. Various general or more detailed references on chemical, biochemical, molecular genetic, cell biological, virological or immunological techniques may be followed. . Sambrook, et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford Univ. Press USA, 1985); Current Protocols in Immunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober 2001 John Wiley & Sons, NY, NY); Real-Time PCR: Current Technology and Applications , Edited by Julie Logan, Kirstin Edwards and Nick Saunders, 2009, Caister Academic Press, Norfolk, UK; Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Guthrie and Fink, Guide to Yeast Genetics and Molecular Biology (Academic Press, New York, 1991); Oligonucleotide Synthesis (N. Gait, Ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, Eds., 1985); Transcription and Translation (B. Hames & S. Higgins, Eds., 1984); Animal Cell Culture (R. Freshney, Ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984); Next-Generation Genome Sequencing (Janitz, 2008 Wiley-VCH); PCR Protocols (Methods in Molecular Biology) (Park, Ed., 3rd Edition) , 2010 Humana Press); Immobilized Cells And Enzymes (IRL Press, 1986); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987) Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and CC Blackwell, eds., 1986); Roitt, Essential Immunology, 6th Edition (Blackwell Scientific Pu blications, Oxford, 1988); Embryonic Stem Cells: Methods and Protocols (Methods in Molecular Biology) (Kurstad Turksen, Ed., 2002); Embryonic Stem Cell Protocols: Volume I: Isolation and Characterization (Methods in Molecular Biology) (Kurstad Turksen Embryonic Stem Cell Protocols: Volume II: Differentiation Models (Methods in Molecular Biology) (Kurstad Turksen, Ed., 2006); Human Embryonic Stem Cell Protocols (Methods in Molecular Biology) (Kursad Turksen Ed., 2006); Mesenchymal Stem Cells: Methods and Protocols (Methods in Molecular Biology) (Darwin J. Prockop, Donald G. Phinney, and Bruce A. Bunnell Eds., 2008); Hematopoietic Stem Cell Protocols (Methods in Molecular Medicine) (Christopher A. Klug, and Craig T. Jordan Eds., 2001); Hematopoietic Stem Cell Protocols (Methods in Molecular Biology) (Kevin D. Bunting Ed., 2008) Neural Stem Cells: Methods and Protocols (Methods in Molecular Biology) (Leslie See P. Weiner Ed., 2008).

Unless specific definitions are given, the nomenclature, tests and techniques used in connection with molecular biology, analytical chemistry, synthetic organic chemistry and medicinal chemistry described herein are those within the skill of the art. It is a known one that is generally used. Also, standard techniques may be used for recombinant techniques, molecular biological synthesis, microbiological synthesis, chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients. good.

Each embodiment described herein applies mutatis mutandis to each of the other embodiments unless stated otherwise.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural unless stated otherwise. Also, in this specification, unless stated otherwise, the term "comprises", as well as its variations, the terms "comprises" or "comprising," understood to include a component or element or component or elements described herein, but exclude a component or element or component or elements other than those described herein not a thing Each embodiment described herein applies mutatis mutandis to each of the other embodiments unless stated otherwise.

Example 1 - Generation of airT cells
We developed a platform for converting normal human CD4 T cells into Treg-like cells to obtain stable recombinant Treg (edTreg; airT) cells by editing the Foxp3 gene (Figs. 1A, 1B, 1C). ,Figure 2). This platform involved generating antigen-specific edTreg cells by introducing the TCR gene using lentivirus.

Antigen-specific T cells were identified by activating PBMC with peptide pools and assessing CD154 expression. In this method, single-cell RNA-seq analysis was used to identify the clonotype of TCRs that are elevated in subjects with type 1 diabetes and to generate the full length of each TCR sequence (Cerosaletti et al. 2017 J. Immunol. PMID: 28566371). Based on the islet-specific TCR sequences identified in this study, several lentiviral TCR constructs were generated to transfer the TCR gene. These TCR constructs expressed the human TCR variable region from the islet-specific TCR and the constant region of the murine TCR, resulting in improved pairing of the human TCR chains when transduced ( Figure 5). Islet-specific TCR expression was verified by T cell proliferation assays using peptides recognized by the TCR (or irrelevant peptides) and antigen presenting cells (APCs). T cells transduced with the islet-specific TCR proliferated only in response to peptides recognized by the TCR and APC (Fig. 6).

To generate antigen-specific airT cells, we edited the Foxp3 locus of CD4 T cells transduced with an islet-specific TCR, and successfully generated airT cells expressing the islet-specific TCR. AirT cells expressing the islet-specific TCR displayed a CD25+, CD127-, CTLA4+ and ICOS+ Treg phenotype (Fig. 7). Remarkably, airT cells expressing the islet-specific TCR exhibit antigen-specific and bystander suppression functions in in vitro suppression assays (FIGS. 7-11). Furthermore, airT cells suppressed the production of inflammatory cytokines such as TNF, IFN-γ, IL-17 and IL-2 by T _eff cells in an airT antigen-specific manner (Fig. 11).

The Treg phenotype, production efficiency and suppressive capacity of airT cells were examined by comparing with expanded nTreg cells. airT cells can increase the number of input PBMCs threefold, but nTreg cells yield only 1-4% of the number of input cells even after 10 days of expansion culture. Furthermore, airT cells displayed a phenotype very similar to nTreg cells, but expressed higher Foxp3, CTLA-4 and ICOS than nTreg cells (Fig. 3).

Remarkably, the suppressive activity of airT cells on proliferation of effector T cells in vitro was equal to or greater than that of expanded nTreg cells (Fig. 4).

Example 2 Acquisition of Treg Phenotype and In Vitro Immunosuppression by FOXP3-Edited Antigen-Specific Human T Cells
Another method for generating FOXP3-edited antigen-specific CD4+ T cells is to isolate, gene-edit and expand antigen-specific effector T cells from healthy subjects or patients with autoimmune disease. developed a method. To investigate whether FOXP3 editing is possible while maintaining proliferation of antigen-specific human T cells, CD4+ T cells obtained from human donors carrying the HLA DRB1*0401 allele were challenged with influenza antigen (flu) and tetanus toxin antigens. Gene editing was performed after expansion in the presence. After gene editing, cells were expanded for an additional 4-7 days in the presence of an antigen cocktail consisting of influenza and tetanus toxoid antigens. The average editing rate (GFP+) at this time was 28±2.1% (FIG. 12). Cells were labeled with a mixture of PE-labeled influenza antigenic peptide-MHC-II tetramer and PE-labeled tetanus toxin antigenic peptide-MHC-II tetramer, and antigen-specific cells were purified by FACS. The resulting tetramer-positive airT (Tmr+airT) cells recapitulated the immunophenotype of activated tTreg cells in which canonical regulatory T cell markers were observed. More specifically, the tetramer-positive airT cells had upregulated expression of FOXP3, CD25, CTLA4 and Helios, and suppressed IL-2 production, unlike the Tmr+mock cells analyzed in parallel. (Fig. 13A). In addition, Tmr+air T cells, unlike Tmr+mock cells, were able to suppress polyclonal activated autologous CD4+ T _eff cells in vitro, indicating that they have an immunosuppressive function (Fig. 13B). ). Based on these results, CD4+ T cells obtained from human peripheral blood can be enriched for target antigen-specific CD4+ T cells by sorting by flow cytometry using tetramers, and gene editing can be used. It was demonstrated that tTreg-like phenotypic properties and suppressive properties that retain antigen specificity can be imparted by modifying with .

This method was further developed to enrich for antigen-specific T cells by stimulating them with model antigens (MP, HA and TT). After approximately two weeks of expansion, the cells were stained with tetramers to identify antigen-specific T cells and edit the FoxP3 locus (Fig. 14). Using this method, antigen-specific Treg cells were generated, and these airT cells exhibited antigen-specific suppressive activity in vitro (Fig. 15). Furthermore, after enrichment of islet-specific T cells by stimulation with a pool of islet-specific peptides, islet-specific T cells with multiple specificities were isolated by tetramer staining. Also in this experiment, islet-specific airT cells were obtained by editing the Foxp3 gene of these cells (Fig. 16, Fig. 17).

Example 3 Generation of Double-Edited Human CD4+ T Cells by Gene Editing of Two Alleles Using Homologous Recombination Repair Figures 18, 19 and 110 show two separate expression cells in the human TRAC locus. It summarizes the experimental methods used to demonstrate whether the cassette can be introduced (Figure 18) and the constructs used in these experiments (Figure 19).

Using CRISPR-based methods, the efficacy of four novel gRNAs targeting the first exon of the human TRAC locus was tested for inducing a complete knockout of the TCR (Figure 20). CD3 expression was assessed by flow cytometry 48 h after RNP delivery, showing that gRNA_1 knocked out 96.8% of CD3 and gRNA_4 knocked out 84.7% of CD3 (Figure 21). ). In addition, on-target site-specific activity was measured by ICE (Inference of CRISPR Edits), and indels were specifically induced by gRNA_1 and gRNA_4 at the TRAC locus compared to predicted off-target sites. was confirmed (Fig. 22). Next, to test the specificity of these novel guides, we evaluated the top three off-target sites of each gRNA (predicted based on bioinformatic analyzes of the most similar sequences in the human genome). These off-target sites were analyzed directly by amplifying each off-target site from nuclease-transfected human T cells. Amplicons were sequenced and analyzed with the ICE program. The observed cleavage activity at each off-target site candidate was 0%. In contrast, on-target sites in the same assay showed 78% cleavage activity by gRNA_1 and 66% cleavage activity by gRNA_4 at the targeted TRAC site (FIG. 23). This result indicated that these new donor templates were highly specific for the TRAC locus.

Next, the dual-editing ability of each gRNA in human CD4+ T cells was examined using a construct that allows easy tracking of successfully edited cells. By generating MND-GFP and MND-BFP cassettes flanked by identical 300 bp homologous arms matched to gRNA_1 or gRNA_4 (Fig. 24A) targeting the TRAC locus, and using these expression cassettes, We tested whether two alleles of the TRAC locus could be edited to generate T cells stably expressing both GFP and BFP. The timeline for cell expansion, gene editing and analysis is shown in Figure 24B. FACS analysis showed that 20.3% of BFP/GFP double-positive cells were obtained with gRNA_1 and 10.6% of BFP/GFP double-positive cells were obtained with gRNA_4. Integration of both repair cassettes was confirmed after a single double-strand break occurred (Fig. 25).

In some cases, selective expansion of recombinant cells in vitro may be useful to obtain sufficient numbers of cells for therapeutic use. To selectively expand double-edited cells, we generated a homologous recombination repair (HDR) knock-in construct containing a split IL-2 chemo-inducible signaling complex (CISC) for enrichment and selection. . A method for enriching gene-edited CD4+ T cells by using IL-2 CISC components in the presence of rapamycin or AP21967 (a rapalog), a rapamycin homolog capable of inducing heterodimerization. reported in the past. For example, see FIG. This method inserts the FRB-IL2RB and FKBP-IL2RG components into the same cassette to allow selection for a single integration event.

To perform this study, the FRB-IL2RB and FKBP-IL2RG components were split into two separate cassettes. By introducing GFP into one cassette and mCherry into the other cassette, two independent integrations could be selected. These constructs are shown in FIG. 26, and the timeline and editing conditions for this experiment are shown in FIG. The initial double-editing rate with these constructs was 1.44% as a percentage of GFP/mCherry double-positive cells (Fig. 28), which was likely due to the large size of the homologous recombination repair template. However, the use of rapalogs could significantly enrich for double-edited cells. The percentage of GFP/mCherry double positive cells increased from 1.4% to 9% in 8 days by treatment with 100 nM rapalog (Fig. 29). Importantly, the proportions of GFP-only positive cells, mCherry-only positive cells, and double-negative cells did not change in the presence of rapalogs, suggesting that dual expression of FRB-IL2RB and FKBP-IL2RG resulted in functional IL- It was suggested that proliferation of GFP/mCherry double-positive cells occurred only in the presence of the 2 CISC protein. As expected, no proliferation was observed in the presence of IL-2 (Figure 30).

Inter-experimental reproducibility and donor-to-donor variability were tested (Figure 31). Cells from the same donor as in the previous experiment (shown in Figure 29) were gene edited and compared to cells from another age-matched Caucasian male donor. Cells from donor R003657 had a double-editing rate of 1.1%, which was similar to results obtained in previous experiments (Figure 29). The compilation rate of the two alleles in the second donor (R003471) was 6.4%. Overall, editing rates varied between donors, but the ratios of GFP-positive, mCherry-positive, and double-positive cells were similar, suggesting that the editing rate variability was consistent with the gene in donor cells. It was suggested that it may be due to how well it can be edited. Importantly, double-edited cells from either donor were enriched in the presence of rapalogs, yielding 13.8% GFP/mCherry double-positive cells in donor R003657 and 28.5% GFP/mCherry in donor R003471. Double positive cells were obtained (Figure 32).

The results of these studies suggest that the double-editing method, which incorporates split-form IL-2 CISCs by homologous-repair-mediated gene editing, provides a means to efficiently select and enrich for double-edited cells, which may be therapeutically effective. It was suggested that it could be used as a method to obtain the required number of edited cells for use.

Results showed that the MND-eGFP-FRB-IL2RB and MND-mCherry-FKBP-IL2RG cassettes were used to successfully edit the two alleles, and that rapalogs could be used to enrich for double-edited cells. Based on the results, constructs capable of introducing FoxP3 in combination with IL-2 CISC components and constructs capable of introducing islet antigen-specific TCR (T1D4) in combination with IL-2 CISC components were generated. and used to generate antigen-specific Foxp3+airT cells (Fig. 33).

Example 4: Targeting Two Alleles to Generate Dual Edited Mouse CD4+ T Cells To Conduct Studies Evaluating the Efficacy of Antigen-Specific FoxP3 air T Cells in Animal Models of Diabetes or Other Autoimmune Diseases , constructed a similar tool for editing the mouse Trac locus. Three novel gRNA target sequences within the first exon of the mouse Trac locus were selected and tested for CD3 knockout in mouse (C57/B6) CD4+ primary T cells (Figure 34). As shown in Figure 35, mCD3 expression was measured by flow cytometric analysis 2 days after transfection, and mTrac_gRNA_2 had the best knockout rate of 87.8%. Similar to constructs for human cells, we generated MND-GFP and MND-BFP constructs to facilitate tracking of gene-edited cells. The constructs used in this experiment and the timeline for this experiment are shown in FIG. As in double-edited human cells that edited the TRAC locus, the double-editing efficiency in mouse cells was relatively low (1.97%) (Figure 37).

Example 5: Function of airT Cells in an Antigen-Specific Mouse Model with Multiple Sclerosis Myelin Oligodendrocyte Glycoprotein Peptide Fragment 35-55 (MOG) Specific to Examine AirT Cell Function in Antigen-Specific In Vivo Conditions T cells for gene editing were selected from the target TCR transgenic mice (C57Bl/6-Tg(Tcra2D2,Tcrb2D2)1Kuch mice (abbreviated as 2D2 mice)). 2D2 transgenic mice develop experimental autoimmune encephalomyelitis (EAE) when challenged with MOG and can be used as a mouse model for multiple sclerosis. EAE in 2D2 mice is not regulated by endogenous 2D2 tTreg cells residing in the central nervous system (CNS), presumably due to the production of abundant proinflammatory cytokines by pathogenic T _eff cells. be done. Adoptive transfer of antigen-specific 2D2 air T cells could suppress proliferation of T _eff cells in the periphery before these activated effector T cells migrate to the central nervous system (Fig. 38). . To test this hypothesis, we designed TALEN and AAV donor templates as reagents that closely mimic the GFP knock-in editing method used to generate GFP+ human airT cells. An improved protocol was designed to stimulate murine T cells for mRNA electroporation, and murine T cells were transfected with mRNAs encoding TALEN pairs specific for the first coding exon of mouse Foxp3. Colony sequencing of PCR-amplified gDNA 7-9 days after transduction showed that approximately 80% of alleles contained indels (FIG. 39). This result indicated that the target site was efficiently cleaved. An AAV donor template was cloned in which mouse Foxp3 homologous arm sequences were substituted for the human Foxp3 homologous arm sequences used in previous homologous recombination repair experiments. This homologous arm was close to the mouse TALEN binding site but did not overlap. mCD4+ T isolated from the spleen and lymph nodes (LN) of 2D2 mice by slightly modifying the conditions used for gene editing of human CD4+ T cells, such as by using AAV5 capsids for introduction of the donor template. Gene editing using cells consistently achieved an editing rate of approximately 25-30% (GFP+ cells). The resulting GFP+ cells had a phenotype of FOXP3+CD25+CTLA-4+ (Fig. 40). For comparison with airT cells generated from 2D2 mice, CD4+ T cells were isolated from littermate mice (C57BL/6) without the 2D2 phenotype and gene-edited to generate polyclonal TCRs with various specificities. AirT cell containing pools were generated. 3.0×10 ⁴ CD4+ T _eff cells from 2D2 mice were then adoptively transferred into lymphopenic Rag1−/− mice along with 3.0×10 ⁴ mock or airT cells. Recipient mice were challenged with adjuvanted MOG35-55 peptide, followed by administration of pertussis toxin to disrupt the blood-brain barrier. Figure 41 shows the experimental timeline including cell transfer, immunization and cell analysis. Recipient mice in this model develop symptoms of EAE (tail and total ptosis followed by hindlimb weakness and paralysis) approximately 7-10 days after cell transfer. To assess effector T cell priming, recipient mice were euthanized 7 days after cell transfer and inguinal and axillary lymph nodes were harvested. The percentage of CD45+CD4+ T cells in the lymph nodes of recipients of antigen-specific 2D2 airT cells was half that of recipients of mock-edited cells and 1.7 times lower than that of recipients of polyclonal C57Bl/6 airT cells. was one-third. The absolute number of CD4+CD45+ T cells was significantly reduced in both airT cell groups compared to mock controls, and the absolute number of CD4+CD45+ T cells in recipients of 2D2 airT cells was 1 and the absolute number of CD4+CD45+ T cells in recipients of C57Bl/6 air T cells was reduced by 18-fold. In both groups, the majority of CD45+CD4+ cells were GFP-, and mice treated with 2D2 edTreg cells had fewer GFP-T cells expressing the inflammatory markers CD25 or IFN-γ (Fig. 42). To determine if the observed effects were due to a decrease in proliferation of T _eff cells, mice were treated with the thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU) 2 hours prior to sacrifice. Aliquots were injected and EdU incorporation into gDNA was detected by flow cytometry using the "Click" reaction (Figure 43). In the group treated with 2D2 airT cells, the overall percentage of GFP- cells that took up EdU was reduced by 22% compared to the group treated with mock-edited cells and 18% compared to the group treated with polyclonal airT cells. Diminished. Importantly, GFP+ cells in the group treated with 2D2 airT cells also incorporated EdU (approximately 25%) and, to a lesser extent, in the group treated with C57Bl/6 airT cells also incorporated EdU. (about 10%). This result was consistent with the ability of tTreg to expand in vivo in response to autoantigen stimulation. These findings indicate that mouse airT cells suppress the priming of pathogenic T _eff cells in vivo, and that antigen-specific airT cells exhibit stronger activity and proliferation than polyclonal airT cells.

Example 6: Function of airT cells in an antigen-specific mouse model of type 1 diabetes The BDC2.5NOD-NSG adoptive transfer model was used to examine the efficacy of antigen-specific airT cells in an in vivo model of autoimmune type 1 diabetes. to determine whether Foxp3-edited antigen-specific T cells can delay or reverse the onset of type 1 diabetes. NOD mice (NOD/ShiLtJ strain) were used as a polygenic model of autoimmune type 1 diabetes. Glycemic development in this model is characterized by moderate glycosuria and non-fasting plasma glucose levels greater than 250 mg/dl. The diabetic mice exhibit hypoinsulinemia and hyperglucagonemia, resulting in selective destruction of pancreatic islet β-cells. This mouse model is currently the most widely used polyclonal autoimmune animal model for the study of spontaneous type 1 diabetes. BDC2.5NOD mice [NOD.Cg-Tg(TcraBDC2.5,TcrbBDC2.5)1Doi/DoiJ] have rearranged TCRα and TCRβ genes derived from the cytotoxic CD4+ T cell clone BDC-2.5 . Mature T cells from this mouse express only the BDC2.5 TCR. This mouse on the NOD background with this TCR transgene has a lower incidence of diabetes compared to NOD/ShiLtJ mice as controls. However, transfer of CD4+CD25-BDC-2.5 T cells into immunodeficient recipient mice rapidly develops marked diabetes. Previous studies have reported that nTreg from antigen-specific BDC2.5 NOD mice can prevent and reverse autoimmune diabetes in NOD mice more effectively than nTreg from polyclonal WT NOD mice. Accordingly, in the present study, using these animal models, Foxp3-edited antigen-specific T cells delayed or reversed the onset of autoimmune type 1 diabetes compared to nTreg from WT NOD mice. I checked if it is possible. First, we tested whether airT cells could be generated in NOD mice. Previous studies have demonstrated that mouse CD4+ T cells from B6 mice can generate airT cells by editing the Foxp3 locus using homologous recombination repair (WO2018/080541 and US Patent Publication 2019/0247443 (these documents are hereby incorporated by reference in their entireties)). The present experiment extends these studies to perform non-homologous end joining (NHEJ) and homologous recombination repair (HDR) using the same AAV donor template in CD4+ T cells from NOD mice. , it was shown that the editing efficiency by these is comparable (Fig. 44). Importantly, Foxp3-edited BDC2.5NOD mouse CD4+ T cells have a Treg phenotype with increased Foxp3 expression and decreased inflammatory cytokine expression compared to mock cells (FIG. 45).

It is conceivable that antigen-specific airT cells can delay or reverse the development of autoimmune type 1 diabetes more effectively than non-antigen-specific airT cells in adoptive transfer NSG mouse models. nTreg cells were also included in this study because nTreg cells have been previously reported to reduce the incidence of type 1 diabetes. The experimental design and phenotypes of transfected T _eff cells, airT cells and nTreg cells are shown in FIG. AirT cells, like nTreg cells, reduced the incidence of diabetes compared to mice administered mock airT cells or T _eff cells alone (Fig. 47). Importantly, administration of BDC airT cells results in a statistically significant reduction in the incidence of diabetes when compared to administration of polyclonal NOD airT cells. This finding indicated that antigen-specific airT cells prevented the development of diabetes more effectively than polyclonal airT cells. Of note, previous studies have shown that the N-terminal GFP-FOXP3 fusion protein functions as a hypomorph and can indeed accelerate autoimmune diabetes in mice on the immunocompetent NOD background. It is Consistent with this observation, in this study also, although antigen-specific nTreg cells performed better than antigen-specific airT cells, a significant suppressive effect was also observed in airT cells expressing the GFP-FOXP3 fusion protein. . Studies with FOXP3-expressing airT cells lacking an N-terminal GFP fusion protein (including airT cells expressing a clinically relevant LNGFR selectable marker in cis configuration; see below) exhibit enhanced protection It is expected to be.

These findings clearly demonstrate that among the BDC2.5 NOD T cells transferred into the adoptively transferred NSG mouse model, antigen-specific airT cells have the ability to prevent the onset of diabetes compared to polyclonal airT cells. rice field.

Example 7 Construction of AAV Donor Template Designs to Generate AirT Cell Products Containing the LNGFR Selectable Marker The Ability to Enrich Mouse Cells After Gene Editing Performs In Vivo Experiments Without Flow Cytometric Sorting This is important when trying to obtain a sufficient number of edited cells for your purposes. To this end, we developed a cis-configured LNGFR selectable marker for use in the purification of mouse edTreg cell products. Figure 48 shows the design of the repair template used to edit mouse Foxp3. Each template contains the LNGF.P2A knock-in (KI), but the type of promoter is different. Furthermore, the difference between the presence and absence of the 0.7 kb UCOE was tested under the control of the MND promoter. When B6 CD4+ T cells were transfected with AAV5 No.1331 (MND-GFP), AAV5 No.3189 (MND-LNGFR) or AAV5 No.3227 (PGK-LNGFR) and RNPs, GFP knock-in editing efficiency and LNGFR The knock-in editing efficiencies of were very similar (Fig. 49, Fig. 50). Furthermore, magnetic field separation using anti-LNGFR microbeads showed an 8.7-fold enrichment of LNGFR+ cells (Fig. 51). These data suggested that LNGFR could be successfully used as a method to select and enrich mouse CD4+ T cells.

Example 8 - Method
Editing Foxp3
CD4+ T cells were isolated from PBMC using the MACS CD4+ T cell isolation kit and treated with CD3/CD28 activated beads (bead:cell=1:1) and IL-2, IL-7 and IL-15. activated with After 48 hours of activation, the beads were removed and the cells rested for 16-24 hours. In Cas9/CRISPR-mediated editing of Foxp3, cells were transfected with an RNP complex consisting of Cas9 and guide RNA by electroporation, followed by transduction with template-containing AAV (AAV FOXP3 exon 1.MND-LNGFRki). did For editing of Foxp3 with TALEN nucleases, cells were transfected with TALEN RNA targeting FOXP3 by electroporation, followed by transduction with template-containing AAV (AAV FOXP3 exon 1.MND-GFPki). . After gene editing, cells were expanded in medium containing IL-2.

Generation of airT cells with islet-specific TCR
CD4+ T cells were isolated from PBMC and activated with CD3/CD28 activated beads and IL-2, IL-7 and IL-15. After 24 hours of activation, transduction with lentiviral vectors encoding TCRs (4.13, T1D2, T1D4, T1D5-1 or T1D5-2) specific for GAD65 or IGRP was performed at MOI=10 using protamine sulfate. gone. Beads were removed after a total of 48 hours of incubation from the initial activation. Gene editing was performed after the cells were rested for 16-24 hours. For Foxp3 editing, the AAV FOXP3 exon 1.MND-LNGFRki template was introduced after electroporation of RNP complexes consisting of Cas9 and guide RNA into cells. After gene editing, cells were expanded in medium containing IL-2, and editing and TCR transduction rates were measured 3-4 days after gene editing. The resulting airT cells were enriched based on LNGFR expression using MACSelect LNGFR beads, aliquoted and cryopreserved until experimental use.

Generation of antigen-specific air T cells To generate T cells specific for influenza antigen (Flu) or tetanus toxoid antigen (TT), CD4+CD25- cells were isolated from PBMCs and incubated with the antigen in the presence of IL-2. Presenting cells (APC) (irradiated autologous CD4-CD25+ cells) were co-cultured with MP, HA and TT peptides. After a total of two 9-day stimulations of CD4+ T cells with these peptides and APC, activation with CD3/CD28 beads and editing of Foxp3 using TALENs and the AAV FOXP3 exon 1.MND-GFPki template was performed. gone. Three days after gene editing, GFP+ cells were sorted by flow cytometry and expanded with stimulation with CD3/CD28 beads. After 7 days of expansion culture, the CD3/CD28 beads were removed, and after further incubation for 4 days, airT cells were harvested and subjected to suppression assay.

To generate islet-specific T cells by stimulation with peptides, CD4+CD25- cells were isolated from PBMC and co-treated with APCs and an islet-specific antigen pool (a total of 9 peptides selected from IGRP, GAD65 and PPI). co-cultured. After 2 weeks of expansion culture, the cells were harvested, stained with tetramers specific for nine pancreatic islet peptides, and sorted by flow cytometry. Selected islet-specific CD4+ T cells were activated with CD3/CD28-activated beads and Foxp3 edited using Cas9/CRISPR and the AAV FOXP3 exon 1.MND-LNGFRki template. Three days after gene editing, cells were stained with each tetramer and analyzed by flow cytometry.

Specific nucleic acid sequences useful in embodiments provided herein are listed in the table shown in FIG.

Example 9 - Comparison of Lentivirally-Delivered FOXP3-Expressing T Cells and airT Cells In vitro and in vivo transfer of a FOXP3 cDNA expression cassette into conventional T cells using lentivirus results in a Treg-like phenotype. and conferred inhibitory properties (Allan et al. Mol Ther 16:194-202 (2008); Passerini et al. Sci Transl Med 5:215ra174 (2013)). To compare with the gene editing method disclosed herein, a lentiviral (LV) construct was generated that delivered a cDNA encoding the same GFP-FOXP3 fusion protein produced by airT cells (Figure 52A). Similar percentages of GFP+FOXP3+ cells were obtained with both gene-editing and viral transduction methods (Fig. 52A). LV-treated cells (LV Tregs) harbored an average of 3.0 copies of lentivirus per genome in GFP+ cells. The airT cells in this experiment had only one targeted insertion per gene-edited T cell from a male donor. Despite the different inserted copy numbers, the MFI of GFP+ cells was consistently lower in LV Treg cells than in airT cells (Fig. 52B), suggesting that expression from genomic rather than cDNA This was consistent with either being more efficient or that loci where LV integrates are poorly transcribed. Both methods of forced expression of FOXP3 resulted in conversion of the T _eff to tTreg phenotype, upregulation of CD25, CTLA-4 and Helios, and downregulation of IL-2, TNF-α and IFN-γ. (Fig. 52C). Except for the expression level of FOXP3, the proportion of cells expressing regulatory T cell markers and the average expression level per cell (assessed by MFI) were very similar between airT cells and LV Treg cells. Furthermore, the growth inhibitory capacity against polyclonal T _eff cells in vitro was comparable between airT cells and LV Treg cells (Fig. 52D). Importantly, however, FACS-purified LV Treg cells showed decreased GFP expression compared to airT cells during the 5-week culture period (Fig. 52E; exceeded 99%). This finding indicates that gene editing utilizing homologous recombination repair can more effectively maintain high expression of FOXP3 than delivery of LV using the same promoter construct. These findings are an unexpected result based on previous reports of FOXP3 delivery using LV, suggesting that editing of the FOXP3 locus using homologous recombination repair permanently induces FOXP3 in CD4 T cells. It supported the concept of becoming a more stable platform for expression.

Example 10 Double Editing Method FIG. 53 shows an overview of the homologous recombination repair gene editing method developed for the purpose of producing antigen-specific airT cells by performing the gene editing method using only homologous recombination repair. indicate. This novel method obviates the need to use LVs for TCR delivery and a) depletes endogenous TCR expression and b) islet-specific T1D TCR (or another antigen-specific TCR associated with disease). ) and c) can be enriched in vitro and in vivo using a novel CISC/DISC IL-2 platform. As shown in Figure 53, a method was developed that could achieve the foregoing objectives by targeting one locus (TRAC) or two loci (TRAC and FOXP3). Examples of successful application of these two strategies are given below.

Figure 54 is a schematic representation of each AAV HDR donor construct used in the studies described below to generate antigen-specific edTreg cells using the double-editing method for one locus or two loci. indicate its number. These AAV HDR donor constructs include those capable of selection by IL2-CISC/DISC and those not capable of selection by IL2-CISC/DISC.

Double Editing of Human CD4+ T Cells—An Example Method for Editing a Single Locus FIGS. 55 and 56 relate to inter-experimental reproducibility and donor-to-donor variability. AAV No. 3207 (MND.GFP.FRB-IL2RG) and AAV No. 3208 (MND.mCherry.FKBP-IL2RG) used in previous experiments were used to edit the genes of cells from two donors and repeat The experimental data were compared with the initial experimental data. In these experiments, all homologous recombination repair templates targeted a single sgRNA cleavage site within the first exon of the TRAC locus. The double-editing rate (incorporation of GFP and mCherry in a split CISC cassette into one cell) in cells from donor R003657 was 2.75% (Fig. 55), which is comparable to the previous two data sets. (Double editing rates for cells from donor R003657 in previous experiments were 1.44% and 1.1%). The double-editing rate in cells from the second donor (R003471) was 6.78% (Figure 56), again similar to the double-editing rate of 6.4% observed in the first dataset. Both donors were Caucasian males of similar age. Overall, there was variation in editing rates among donors, but when looking at each donor, experiments showed similar rates of editing, suggesting that this variation in editing rates reflects how much of the gene in the donor's cells has changed. It was suggested that this was due to the ability to edit well and not due to variability between different experiments on the same donor. Importantly, the results observed in previous experiments in the presence of a rapamycin analog (rapalog, AP21967) capable of inducing heterodimerization when using edited T cells from any human donor. were able to enrich for double-edited cells with similar efficiency. In this study, enrichment for 7 days using rapalogs enriched GFP/mCherry double positive cells from donor R003657 by 44.7% and GFP/mCherry double positive cells from donor R003471 by 46.1%. (Fig. 55, Fig. 56). As expected, it was not enriched in the presence of IL-2.

The results of these studies suggest that the integration of split IL-2 CISCs by homologous recombination repair-mediated double-editing provides a means for efficient selection and enrichment of double-edited cells, providing both donor-to-donor and It was shown to be reproducible between replicate experiments. Results of successful double-editing using MND.GFP.FRB.IL2RB cassette (No.3207) and MND.mCherry.FKBP.IL2RG cassette (No.3208) and double-editing cells using rapalog Based on the results that we were able to enrich A construct (No. 3243) capable of introducing an antigen-specific TCR (T1D4) was prepared and used to generate antigen-specific FOXP3+edTreg cells (Fig. 57).

Using the MND.T1D4.FRB.IL2RB construct (No.3243) replacing GFP with the T1D4 TCR and the MND.HA.FOXP3.FKBP.IL2RG construct (No.3240) replacing mCherry with HA-tagged FOXP3 , tested the initial editing rate and proliferation of T1D4-positive human edTreg cells expressing FOXP3. These constructs are shown in Figure 57(A), and the timeline and editing conditions for this experiment are shown in Figure 57(B). The percentages of GFP/mCherry double-positive cells using CISC constructs containing GFP and CISC constructs containing mCherry were 2.75% (Fig. 55) and 6.37% (Fig. 56). The percentage of /T1D4 double-positive cells was as low as 0.65% (Fig. 57). Thus, the FOXP3/T1D4 double-positive cells could be significantly enriched despite the low initial editing rate. Treatment with rapalogs for 8 days enriched FOXP3/T1D4 double-positive cells from 0.65% to 11%, whereas treatment with IL-2 enriched only 1.1% (Fig. 57 (C)). The lower initial editing rate with these constructs than with the split CISC construct containing GFP (No. 3207) and the split CISC construct containing mCherry (No. 3208) was attributed to the T1D4 TCR. It is possible that the size of the homologous recombination repair template contained was large. The size of MND.T1D4.FRB.IL2RB (No.3243) was 4.3 kb, which was significantly larger than MND.mCherry.FKBP.IL2RB (No.3208) with a size of 2.7 kb.

Improving the editing rate and/or increasing the enrichment rate of FOXP3/TCR double positive cells may be important to obtain sufficient numbers of edited cells for therapeutic use. To improve the initial editing efficiency, conditions were modified with different serum concentrations during the gene-editing step. Figures 58 and 59 show the results of double-edit experiments using MND.HA.FOXP3.FKBP.IL2RG (No.3240) and MND.T1D4.FRB.IL2RB (No.3243) as AAV constructs. , which compares four serum concentrations in the gene-editing stage of human CD4+ T cells. When analyzed by FACS, the initial edit rate improved from 1.8% with 20% FBS to 3.96%-4.75% with low or no serum (Fig. 58). Importantly, when cells recovered in medium containing 2.5% FBS were enriched by treatment with IL-2 for 7 days, the percentage of double-positive cells was 1.97%, whereas rapalog The percentage of double-positive cells increased from 1.8% to 17.6% when enriched with treatment, and double-positive cells were enriched nearly 10-fold (Figure 59).

Taken together, the results shown in Figures 55-59 demonstrate that double editing using homologous recombination repair can be efficiently performed at the TRAC locus and can be enriched using the IL-2 CISC platform. It was shown that antigen-specific airT cells can be obtained.

Double editing of human CD4+ T cells - an example of how to edit two loci
As another dual-editing strategy for generating antigen-specific airT cell products containing IL-2 CISC components, we developed a construct targeting the TRAC locus and a construct targeting the FOXP3 locus, Editing of the locus was performed. As shown in the schematic of Figure 53, instead of using two constructs targeting the TRAC locus, one construct targeting the TRAC locus and one targeting the FOXP3 locus are used. In this method, the double editing rate can be improved, and it is thought that the multiple methods developed in the previous experiment for sustained expression of FOXP3 can be used in combination. To test this method, we developed a construct that allows easy tracking of successfully edited cells. IL-2 was linked to CISC by using the MND.mCherry.FKBP.IL2RG (No.3251) cassette with FOXP3 homology arms and the MND.GFP.FKB.IL2RB (No.3207) cassette with TRAC homology arms. We tested whether it is possible to generate double-edited cells that stably express both mCherry and GFP. The constructs used and the timeline for gene editing, cell expansion and analysis are shown in FIG. Since editing of one locus in the previous experiment suggested that the double-editing rate was higher at lower serum concentrations (Fig. 58), in this experiment, editing conditions with high serum concentration versus low serum Editing conditions according to density were compared. FACS analysis showed that the method of editing two loci was successful in both serum concentration conditions (Fig. 61). Similar to single-locus editing, the use of media containing 2.5% serum in the gene-editing stage significantly improved the double-editing rate at day 3 (when media containing 20% serum was used). was 4.99% compared to 11.0% when medium containing 2.5% serum was used). Edited cells in media containing 2.5% serum were then expanded in the presence of IL2 or rapalog (AP21967) for 10 days. Figure 62 shows that mCherry/GFP double positive cells were highly enriched in the presence of rapalogs, reaching a total of 59%.

To test inter-experimental reproducibility and to further improve the initial edit rate, we explored a variety of additional editing conditions. FIG. 63 shows a timeline of gene editing, cell expansion and analysis and a summary of each editing condition. In this experiment, medium containing 2.5% serum was used in the gene editing step in all conditions. Editing results under each condition using AAV No. 3207 and AAV No. 3251 in the presence and absence of the homologous recombination repair enhancer (HDR-E), varying the virus ratio (volume %) for each reaction. compared. Figure 64 shows histograms summarizing the flow cytometry data for each condition 3 days after compilation. Editing rates in medium containing 2.5% serum in this experiment were comparable to the previous experiment shown in Figure 61 (the percentage of GFP/mCherry double positive cells in this experiment was 15.4%). whereas the percentage of GFP/mCherry double-positive cells in the previous experiment was 11%). Furthermore, when the double-edited cell population was enriched with rapalogs, the proportion of GFP/mCherry double-positive cells reached 54% (Fig. 65), a result comparable to previous experimental data, suggesting that the inter-experimental was shown to be reproducible. Although the presence of HDR-E did not affect the initial editing rate, the proportion of virus in the reaction affected the editing outcome (Figure 64). The results suggest that 10% of each virus to medium volume is optimal compared to 15% of each virus to medium volume or 30% of both viruses combined. It was shown that there is

As a result of these studies, by using the double-editing method of editing two loci disclosed herein, a split IL-2 CISC cassette was introduced to efficiently generate double-edited cells with rapalogs. It was shown that it can be concentrated to The success of this method of generating double-edited cells and enriching them led to constructs targeting the FOXP3 locus to express FOXP3 together with IL-2 CISC components and a construct targeting the TRAC locus. designed and cloned constructs expressing islet antigen-specific TCR (T1D4) together with IL-2 CISC components. Using these HDR donors and various other HDR donors, antigen-specific FOXP3 airT cells can be generated (Figure 66).

In-frame knock-in to the TRAC locus as a double-editing method As a further modified or improved method of the double-editing method by the present inventors, the first exon of the TRAC locus is targeted and a promoter is added. A method was established to knock-in in-frame a TCR cassette containing the IL-2 CISC component that lacks the IL-2 CISC component (Fig. 67). This method of editing exploits the promoter/enhancer activity of the endogenous TRAC locus to induce expression of antigen-specific TCRs. The advantage of this method is that it eliminates the expression of the endogenous TCR (and thus the possibility of inappropriate pairing with the delivered TCR components) and concomitant can induce near-endogenous levels of autoantigen-specific TCR expression. To establish this method, a proof-of-concept mCherry IL-2 CISC-containing construct (No. 3253) was used to test gene editing and exogenous gene expression (FIG. 68). The percentage of mCherry-positive cells lacking CD3 was 63.8% (when only AAV was introduced, the percentage of CD3-expressing cells was 99.9%, but AAV containing P2A.mCherry.FRB.IL2RB (No. 3253), the percentage of CD3-expressing cells decreased to 3.01%). After gene editing, mCherry expression could be readily detected by flow cytometry. As expected, the MFI of mCherry expression in cells edited with AAV containing P2A.mCherry.FRB.IL2RB (No.3253) showed the MND promoter expression cassette (MND.mCherry.FKBP.IL2RG (No.3208)). It was lower than the T cells that were used to edit the same locus (Fig. 69). Thus, this method of homologous recombination repair could exploit the promoter/enhancer activity of the endogenous TRAC locus to drive transgene expression.

In follow-up studies, two HDR donor cassettes will be used to double edit the TRAC locus (and utilize the endogenous promoter) and/or edit both the TRAC and FOXP3 loci. Each HDR donor introduces a split component of IL-2 CISC to enable enrichment of dual-edited cells and delivers an antigen-specific TCR (under the control of the promoter of the TRAC locus), Furthermore, FOXP3 is expressed through expression from cDNA or by targeting expression of endogenous FOXP3. A dual-editing approach that edits two loci, a split CISC mCherry construct (P2A.mCherry.FRB.IL2RB (No.3253)) that utilizes the endogenous promoter of the TRAC locus and edits the FOXP3 locus. Test in combination with MND.GFP.FKBP.IL2RG (No.3273) for Expression of the two components of the split IL-2 CISC from two different promoters may affect the overall function of CISC, suggesting that P2A.mCherry.FRB.IL2RB (No.3253 ) and P2A.GFP.FKBP.IL2RG (No.3292) to induce the expression of each component of IL-2 CISC from the endogenous promoter of the TRAC locus. Editing methods are also tested (Fig. 70).

Given the successful editing of the two alleles (by using one or both promoterless mCherry/GFP constructs and enriching for double-edited cells with rapalogs), antigen-specific FOXP3+ airT cells As an alternative method for making , constructs are made that can introduce FOXP3 and an antigen-specific TCR (T1D4) in combination with IL-2 CISC components (see Figure 70). AirT cells generated using these methods are compared to cells in which an antigen-specific TCR is induced from an exogenous MND promoter.

Decoy - double editing using the CISC (Split DISC) construct
CISC-expressing cells expand efficiently in the presence of a rapalog (AP21967) capable of inducing heterodimerization, although the FDA-approved drug rapamycin may be preferred for clinical applications. It is well established that binding of rapamycin to its target, mTOR, in the cell exerts an inhibitory effect on T cell proliferation. To address this issue, constructs and methods utilizing naked "decoy" FRB domains expressed inside CISC-expressing cells are disclosed in WO2019210057, which is incorporated herein by reference in its entirety. cited). Constructs expressing naked FRB domains with CISC are referred to as "decoy-CISC" or "DISC."

To confirm whether DISC functions in the double-editing method, CISC-containing constructs were modified to include an additional naked FRB domain at the 3' end of the CISC receptor (Figs. 70, 71). Constructs containing additional FRB domains along with the CISC component are referred to as "decoy-CISC" or "DISC." When this construct is used in a double-editing method, it is called a "split-type DISC". When utilizing a split DISC, the naked FRB domain competes with the endogenous FRB domain of mTOR for binding to rapamycin, resulting in rapamycin-mediated binding by split CISC components without inhibiting cell proliferation. Signal transduction can be induced.

As shown in Figure 71, double-editing T cells with a mCherry CISC construct (mCherry DISC, No. 3280) and a GFP CISC construct (No. 3207) containing an additional FRB domain resulted in 7.07% GFP/mCherry Double positive cells were obtained. As expected from this experiment with the DISC construct, GFP/mCherry double-positive cells were enriched to a similar degree (79.5% and 86.4%, respectively) by treatment with rapamycin or rapalog (AP21967) (Fig. 72).

Experiments are performed to examine the in vivo enrichment/engraftment of cells edited with the GFP/mCherry split DISC construct in NSG mice treated with rapamycin. GFP/mCherry double edited cells have improved engraftment/enrichment in vivo. By using a FOXP3-containing construct and a T1D4-containing construct, we extend this study to assess engraftment and expansion of islet-specific airT cells in vivo. The DISC construct (MND.FOXP3.FKBP.IL2RG.FRB (No.3262)) shown in Figure 73 was combined with the MND.T1D4.FRB.IL2RB construct (No.3243) prepared earlier to form Double editing is performed to generate islet-specific airT cells that can be enriched in vivo using rapamycin.

Example 11 - Functional activity of antigen-specific mouse airT cells in vitro and in vivo
Characterization of mouse airT cell products in vitro:
Studies were designed to identify advantageous HDR donor template designs in generating airT cell products with a suppressive Treg-like phenotype from murine CD4+ T cells. The questions to be addressed in this study include: a) Which of the tested promoter/enhancer constructs is able to provide the best performing airT cells in vitro and ultimately in vivo? and b) the use of a clinically relevant cis-configured LNGFR selectable marker raises the question of whether airT cells can be enriched in a manner suitable for GMP-compliant manufacturing. The question was whether it was possible. Figures 74 to 80 show (1) the results of experiments evaluating the use of the clinically relevant cis-configured LNGFR selectable marker as a method for enriching mouse cells after gene editing; In addition) results of experiments testing various promoter candidates, (3) results of experiments testing two Foxp3 homologous arms of different sizes in the donor template, and (4) results of promoters introduced within the Foxp3 locus. Results of experiments comparing donor templates containing UCOE elements with donor templates without UCOE elements (as a means of limiting silencing) are shown.

First, the effect of extending the homologous arms of the MND.LNGFR.P2A donor template of the Foxp3 gene from 0.6 kb to 1.0 kb on the editing efficiency of CD4+ T cells from C57BL/6 mice was evaluated (FIG. 76). The results of this study show that MND.LNGFR.P2A (No.3261), which contains a 1.0 kb homologous arm, has slightly higher editing efficiency than MND.LNGFR.P2A (No.3189), which contains a 0.6 kb homologous arm. It has been shown. Extension of this homologous arm increased editing efficiency by approximately 10%, and this improvement in editing efficiency was reproducible between experiments, making it desirable for the generation of mouse MND.LNGFR.P2A airT cells in subsequent studies. AAV No.3261 was selected as the target donor template. We also tested the editing efficiency and post-enrichment purity of C57BL/6 edTreg cells using AAV donor templates containing different promoters (FIG. 76). As a result, the overall editing rate and purity of LNGFR+ cells after enrichment were similar among AAV donor templates containing the MND promoter, the PGK promoter, and the EF-1α promoter. It was shown that the Furthermore, the purities of LNGFR+ and GFP+ cells after enrichment were also comparable, indicating that LNGFR can be used as a method to select and enrich mouse CD4+ T cells.

Importantly, although the editing efficiency and the purity of edited cells were similar when using various AAV donor templates with different promoters, the level of FOXP3 expression varied with the type of promoter. Figure 77 shows GFP in mock-edited CD4 + T cells of C57BL/6 mice, edited with MND.GFP.KI (No.1331), or edited with PGK.GFP.KI (No.3209) and the results of evaluating the expression of FOXP3. This result indicated that the MFI of FOXP3 in cells edited with MND.GFP.KI (No.1331) was significantly higher than in cells edited with PGK.GFP.KI (No.3209). Expression of FOXP3 in PGK.GFP.KI (No. 3209)-edited CD4+ cells was comparable to that observed in splenic nTreg cells. These studies showed that introduction of various promoters into the endogenous Foxp3 locus could control the overall expression of FOXP3 in airT cell products. From this result, it is considered that airT cell products can be produced flexibly, and airT cells with various functional properties can be obtained. The relationship between FOXP3 expression level and function in vitro or in vivo was further investigated in the studies presented in Figures 79 and 85 (see below). We also tested whether the ubiquitous chromatin opening element (UCOE) could stabilize the expression of FOXP3 (Fig. 77). UCOE can limit its adverse effects on promoter elements by suppressing silencing. These studies showed that FOXP3 was stable in the presence or absence of UCOE, and that the inclusion of the UCOE element did not adversely affect the relative expression of FOXP3 (MND.GFP.KI (No .1331) and MND.GFP.KI (No.3213) with UCOE). This result suggests that donors whose silencing is suppressed by the inclusion of UCOE perform effectively, and that the inclusion of UCOE can protect expression in vivo or over time. , could improve the duration of functional activity, suggesting that UCOE may be useful in airT cell products.

Further studies were performed to (a) assess the functional activity of LNGFR-expressing airT cells in vitro and (b) the promoter driving the expression of endogenous FOXP3 had some effect on the functional activity of Treg cells. We investigated whether the MND.GFP.KI-edited airT cells and MND.LNGFR.P2A-edited airT cells were sorted by flow cytometry and in the presence and absence of these airT cells, the in vitro results outlined in FIG. Proliferation of T _eff cells was analyzed by suppression assay. The effects of these airT cells were compared with the activity of purified mouse nTreg cells. From the results shown in Figure 79, both mouse airT cells (generated using MND.GFP.KI HDR donors or MND.LNGFR.P2A HDR donors) and nTreg cells exhibited potent suppressive function in vitro. It was shown that the inhibitory function was comparable. Furthermore, these findings suggest that airT cells expressing the LNGFR selectable marker (cells edited with MND.LNGFR.P2A (No.3261)) can be used as a method to select and enhance mouse CD4+ cells without loss of functional activity. It was successfully used and shown to be useful in in vitro or in vivo modeling of the functional activity of human airT cell products utilizing this clinically relevant selectable marker.

Using the same in vitro suppression assay, we investigated whether the level of expression of FOXP3 (assessed in Figure 77) correlates with the functional activity of the promoter by examining the functional activity of promoters of varying strength. Figure 80 shows the LNGFR construct using the MND promoter (MND.LNGFR.P2A), the LNGFR construct using the PGK promoter (PGK.LNGFR.P2A), or the LNGFR construct using the EF-1α promoter (EF-1a.LNGFR. P2A) shows a comparison of C57BL/6 mouse-derived CD4+ T cells edited with MND.GFP.KI, C57BL/6 mouse-derived CD4+ T cells edited with MND.GFP.KI, and C57BL/6 mouse-derived nTreg cells. The results of this experiment showed that mouse airT cells with the MND promoter exhibited similar suppressive function to nTreg cells. In contrast to constructs containing the MND promoter, airT cells with the PGK promoter showed only partial repressive function in vitro and airT cells with the EF-1α promoter showed no repressive function. Interestingly, FOXP3 expression in PGK.GFP.KI-edited cells was comparable to nTreg cells (Fig. 77), whereas the in vitro suppressive activity of nTreg cells was significantly higher than cells. These findings lead to the surprising conclusion that a threshold level of FOXP3 expression in edited CD4+ T cells would need to be exceeded for proper reprogramming and effective functional activity in vitro. It was suggested. The results described below also clearly demonstrate that the MND promoter was effective in reducing diabetes in vivo.

Functional characterization of edTreg cell products in vivo:
The experimental results summarized above demonstrate that mouse airT cells harboring a clinically relevant cis-configured LNGFR selectable marker retained functional activity in vitro, and that the MND promoter was more repressible than other promoters in vitro. It was suggested that it had activity. Extending these studies, we tested islet-specific airT cell products in an NSG adoptive transfer diabetes model in which diabetes was rapidly developed by transfer of islet-specific NOD (mouse) CD4+ T cells into adult NSG recipient mice. evaluated.

Using this model, whether islet-specific MND.LNGFR.P2A airT cells derived from NOD BDC2.5 mice can delay or prevent the onset of diabetes. evaluated what The in vitro experiments shown in Figures 76-80 utilized cells enriched by flow cytometric sorting, but such a sorting process is time consuming and expensive. In addition, the FACS sorting step can significantly affect the engraftment and/or survival of adoptively transferred cells in vivo. To efficiently enrich for gene-edited mouse airT cells that can be used in vivo, we compared the purification of airT cells purified using different methods and their functional activity. Specifically, (1) enrichment of LNGFR+ cells by cell sorting using a flow cytometer and (2) enrichment of LNGFR+ cells using separation on an LNGFR column were compared. Figures 82-83 show flow cytometry plots before and after purification using FACS sorting or column enrichment. Although the LNGFR+ cell population obtained by purification using the FACS sorting method was somewhat purer, column enrichment also yielded a ~84% pure LNGFR+ cell population, significantly saving time and materials. I was able to Importantly, delay or prevention of diabetes onset was observed in both LNGFR+airT cells purified by column enrichment and by FACS sorting (FIG. 84).

The combined data shown in Figures 82-84 demonstrated that both LNGFR column separation and FACS sorting yielded highly purified islet-specific edited cells. Both cell products were shown to have a Treg-like phenotype, as they highly expressed LNGFR/FOXP3 and reduced or prevented diabetes in vivo.

Furthermore, Figure 85 shows that the functional activity of islet-specific airT cell products in the NSG adoptive transfer model varied depending on the promoter used to induce endogenous FOXP3. Both MND.GFP.KI-edited cells and nTreg cells were able to delay or prevent the onset of diabetes, whereas PGK.GFP.KI-edited airT cells also delayed the onset of diabetes. could not be prevented. This result was consistent with the in vitro repression data presented in Figure 80, suggesting that promoter selection plays a role in obtaining optimized function. Importantly, consistent with the protective effect seen against diabetes, islet-specific airT cells were recruited to the pancreas and remained viable in the NSG model while stably expressing FOXP3 (Fig. 86).

These data indicated that mouse airT cells that can be used for in vitro and in vivo studies can be generated from T _eff cells. Consistent with findings in human T cells, use of the MND promoter in mouse T _eff cells led to airT cells that highly expressed FOXP3 and exerted in vitro suppressive activity as potent as nTreg cells. could be effectively converted to Importantly, islet-specific mouse airT cells and nTreg cells with the MND promoter (1) exert comparably potent suppressive functions in vitro and (2) are suppressed by islet-specific T _eff cells in recipient mice. Prevented diabetes mellitus. Furthermore, from the above data, (3) airT cells expressing the LNGFR selectable marker can be enriched in vitro and function in vivo without loss of functional activity, and (4) airT cells with the MND promoter are , showed better performance than airT cells generated with other promoters such as PGK and EF1α, indicating that the choice of promoter plays a role in improving function.

The NSG adoptive transfer diabetes model described herein enables us to explore important functional characteristics of murine airT cells, such as trafficking to lymph nodes, cell proliferation, activation state, and restrictive capacity for initial activation of T _eff cells. We were able to evaluate quickly. This method was used to compare the functional activity of enriched antigen-specific LNGFR airT cells in an immunocompetent NOD background type 1 diabetic mouse model.

Editing the Rosa26 locus to generate gene-edited mouse T cells To extend the toolset for evaluating the efficacy of antigen-specific FOXP3 air T cells in animal models of diabetes or other autoimmune diseases, A gRNA targeting the mouse Rosa26 locus was designed and tested. The Rosa26 locus is a well-characterized safe harbor locus that has been used in previous studies to stably express transgenes integrated into mouse models. We selected two novel gRNA target sequences within the intron region of the Rosa26 locus, which are in the vicinity of previously reported gRNA target sites, to deliver RNPs to primary mouse CD4+ T cells, resulting in ICE (Inference of On-target site-specific activity was measured by CRISPR Edits). ICE analysis confirmed the induction of R26_gRNA_1-specific indels at the Rosa26 locus (Fig. 87).

We then tested the editing ability of each gRNA in mouse T cells using constructs that appeared to allow easy tracking of successfully edited cells. A MND-GFP cassette flanked by 300 base pairs of identical Rosa26 homologous arms adapted to R26_gRNA_1 (No. 3245) was generated, and this MND-GFP cassette was used to edit the Rosa26 locus to generate GFP. Stably expressing T cells were generated. The timeline for cell expansion, gene editing and analysis is shown in FIG. From the results of FACS analysis, 3 days after gene editing, the ratio of GFP-highly expressing cells was 0.02% when AAV No.3245 alone was introduced, whereas AAV No.3245 and RNP were introduced. It was shown that the percentage of cells with high GFP expression was 11.4% in this case, confirming that the MND-GFP repair cassette was integrated into the Rosa26 locus (Fig. 89). FACS analysis performed 8 days after gene editing showed that a similar proportion of GFP+ cells (10.8%) was obtained, indicating stable expression of GFP (Fig. 90).

We were able to edit the Rosa26 locus in murine T cells by using the MND-GFP cassette, which allowed us to construct the mFoxp3 coding sequence with the LNGFR marker (for purification) and another candidate promoter (besides the MND promoter). A repair template was constructed to generate another construct capable of stably expressing FOXP3 at this safe harbor locus in mouse cells (Figure 91). These constructs are used to investigate double editing in mouse cells with the aim of generating antigen-specific FOXP3-expressing mouse airT cells for use in autoimmune disease mouse models. FOXP3 mutational variants predicted to have improved stability are tested. This FOXP3 mutant is a 4×CDK phosphorylation mutant in which proteolysis-associated phosphorylation is prevented by substitution of alanines for a series of four target residues phosphorylated by cyclin-dependent kinases. is included.

These data indicate that the Rosa26 locus, a safe harbor site, can be used for gene editing of mouse T cells using homologous recombination repair. This finding enables dual-editing studies in mouse T cells, and concurrent studies in human T cells, facilitating the creation of non-clinical animal models of antigen-specific airT cells.

Development of mouse cell expansion tools using CISC elements A key feature of the antigen-specific human airT cell platform is the use of, for example, the IL-2-CISC system to expand airT cells in vitro and in vivo. It can be cultured. To assess the function of airT cells containing the IL-2-CISC cassette in immunocompetent animal disease models, human IL-2R sequences containing the CISC/DISC cassette were induced in vitro and in vivo in the presence of rapalogs/rapamycin. Experiments were conducted to investigate whether selective expansion and proliferation of mouse cells could be promoted. Experimental data using a lentiviral construct (No. 1272) containing the mCherry reporter driven by the MND promoter and the human IL-2 CISC element in cis configuration demonstrated this concept. Figure 92 shows a schematic of the lentiviral cassette and the timeline for T cell transduction, expansion and analysis. In this study, two days after transduction, transduced cells were cultured in the presence of (a) IL-2, IL-7 and IL-15 or (b) rapalog alone. , (c) cultured in the presence of rapalogs and boosting with CD3/CD28 beads. Figure 93 shows that 8.85% of transduced cells expressed mCherry and were further enriched by treatment with rapalog for 3 days. The enrichment was greatest in transduced T cells co-treated with rapalogs and boosted with CD3/CD28 beads (46.1%).

These data demonstrate that IL-2 CISC technology can be used to enrich mouse CD4+ T cells and that human CISCs are functional in the mouse system. These findings indicate that studies investigating the enrichment of antigen-specific mouse airT cells and their function can be carried out using split-CISC or split-DISC methods in nonclinical animal models.

Example 12 - Generation of antigen-specific edTreg cells and testing thereof
Rheumatoid arthritis antigen-specific TCRs identified from rheumatoid arthritis patients
A rheumatoid arthritis antigen-specific TCR was identified from a T cell clone isolated from a rheumatoid arthritis patient. Based on the sequences of these TCRs, several lentiviral TCR constructs were generated for transferring the TCR gene. Table 1 shows the rheumatoid arthritis antigen-specific TCR-encoding lentiviral constructs generated in this study, their epitope specificity and HLA-restriction. Targeted T-cell epitope sequences included citrulline modifications. TCRs recognizing citrullinated vimentin, citrullinated aggrecan, citrullinated CILP or citrullinated enolase were identified from T cell clones previously isolated from rheumatoid arthritis patients.

CD4+ T cells were isolated, activated with CD3/CD28 beads, and transduced with a lentivirus encoding a rheumatoid arthritis antigen-specific TCR. Flow cytometry plots of mTCRb expression gated on CD4+ cells 9 days after transduction are shown (Fig. 117A). CD4+ T cells transduced with TCRs specific for rheumatoid arthritis antigens were labeled with CTV and co-cultured with APCs (irradiated PBMCs) in the presence of peptides recognized by the TCR or DMSO for 3 days. Flow cytometry plots show cell proliferation as a function of CTV dilution (Fig. 117B). Expression of rheumatoid arthritis-specific TCR was verified by a T cell proliferation assay using peptides recognized by the TCR and antigen presenting cells (APC). T cells transduced with rheumatoid arthritis-specific TCRs (vimentin-, aggrecan-, CILP- or enolase-specific TCRs) proliferated in response to peptides recognized by the TCR and APC.

Suppressive Activity of Enolase-Specific edTreg Cells Antigen-specific Treg cells were generated by editing the Foxp3 locus in CD4 T cells transduced with an enolase-specific TCR. This resulted in enolase-specific edTreg cells. FIG. 118A. Flow cytometry showing mTCRb and LNGFR/Foxp3 expression at day 7 in non-lentivirally transduced edited cells (Untd Edited) and edited cells expressing Enol326-TCR (Enol326 Edited). A metric plot is shown. At day 10, edTreg cells were enriched by expression of LNGFR. LNGFR- cells were used as mock cells for suppression assays. The transduced Enol326-TCR had specificity for an epitope consisting of residues 326-340 of enolase.

FIG. 118B shows polyclonal and antigen-specific suppression assays using enolase-specific edTreg cells. Enolase-specific Teff cells were generated by transducing CD4+ T cells with lentivirus encoding Enol326-TCR and expanded for 15 days. For polyclonal suppression assays, Enol326 Teff cells were either incubated without added Treg cells or transduced in the presence of anti-CD3/CD28 beads added at a bead:cell ratio of 1:30. Enol326 Teff cells were incubated with untd edTreg cells, Enol326 edTreg cells or mock cells. Antigen-specific suppression assays co-cultured Enol326 Teff cells with APCs and Enol326 peptide in the absence of Treg cells or in the presence of untransduced (untd) edTreg cells, Enol326 edTreg cells or mock cells. . In both suppression assay setups, Teff cells were labeled with CTV, edTreg or mock cells were labeled with EF670, and Teff and edTreg or mock cells were co-cultured at a 1:1 ratio. After 4 days of co-cultivation, cells were stained and proliferation of Teff cells was analyzed from CTV dilutions. Figure 118C shows the rate of inhibition of Teff cell growth by no Treg cells, untdd edTreg cells, Enol edTreg cells or mock cells in the presence of anti-CD3/CD28 (black) or APC and enolase peptide (grey). FIG. 118B shows a graph showing results calculated from the proliferation rate shown in FIG. 118B. In these in vitro suppression assays, enolase-specific edTreg cells exhibited antigen-specific and polyclonal suppressive functions against antigen-specific effector T cells.

Suppressive activity of edTreg cells specific to CILP
Suppressive activity of CILP-specific edTreg cells was measured. FIG. 119A shows non-transduced cells by gating on LNGFR+Foxp3+ in edited cells that were not lentivirally transduced or transduced with lentivirus encoding CILP297-1-TCR. Flow cytometry plots analyzing mTCRb expression in edTreg or CILP297-1 edTreg cells are shown. The CILP297-1 TCR had specificity for an epitope consisting of residues 297-311 of CILP. FIG. 119B shows polyclonal and antigen-specific suppression assays using CILP-specific edTreg cells. CILP-specific Teff cells were generated by transducing CD4+ T cells with lentivirus encoding CILP297-1-TCR and expanded for 15 days. For polyclonal suppression assays, CILP Teff cells were incubated without Treg cells, or untransduced (untd) edTreg cells, CILP edTreg cells, or mock cells were added in the presence of anti-CD3/CD28 beads. were incubated with CILP Teff cells. For antigen-specific suppression assays, CILP Teff cells, APCs and CILP297 peptide were co-cultured in the absence of Treg cells or in the presence of untransduced (untd) edTreg cells, CILP edTreg cells or mock cells. . FIG. 119C shows the growth inhibition rate of CILP Teff cells by no Treg cells, untd edTreg cells, CILP edTreg cells or mock cells in the presence of anti-CD3/CD28 (black) or APC and CILP peptide (grey). 119B shows a graph showing the results calculated from the proliferation rate shown in FIG. 119B. Similar results were obtained using CILP-specific edTreg cells. In these in vitro suppression assays, CILP-specific edTreg cells exhibited antigen-specific and polyclonal suppressive functions against antigen-specific effector T cells.

Suppressive activity of vimentin-specific edTreg cells Suppressive activity of vimentin-specific edTreg cells was measured. Figure 120A shows non-transduced edTreg cells by gating on LNGFR+Foxp3+ edited cells that were not lentivirally transduced or transduced with lentivirus encoding Vim418-TCR. or flow cytometry plots analyzing the expression of mTCRb in Vim418 edTreg cells. The Vim418 TCR had specificity for an epitope consisting of residues 418-431 of vimentin.

FIG. 120B shows polyclonal and antigen-specific suppression assays using vimentin-specific edTreg cells. Vimentin-specific Teff cells were generated by transducing CD4+ T cells with lentivirus encoding the Vim418 TCR and expanded for 15 days. For polyclonal suppression assays, Vim Teff cells were incubated without Treg cells, or untransduced (untd) edTreg cells, Vim edTreg cells, or mock cells were added in the presence of anti-CD3/CD28 beads. was incubated with Vim Teff cells. For antigen-specific suppression assays, Vim Teff cells, APCs and Vim418 peptide were co-cultured in the absence of Treg cells or in the presence of untd edTreg cells, Vim edTreg cells or mock cells. Figure 120C shows the growth inhibition rate of Vim Teff cells by no Treg cells, untd edTreg cells, Vim edTreg cells or mock cells in the presence of anti-CD3/CD28 (black) or APC and vimentin peptide (grey). 120B shows a graph showing the results calculated from the proliferation rate shown in FIG. 120B. In these in vitro suppression assays, vimentin-specific edTreg cells exhibited antigen-specific and polyclonal suppressive functions against antigen-specific effector T cells.

Antigen-specific suppression of aggrecan-specific Teff cells and bystander suppression effect By using aggrecan-specific edTreg cells, antigen-specific suppression of aggrecan-specific Teff cells was demonstrated, and vimentin-specific edTregs By using cells, a bystander suppression effect on aggrecan-specific Teff cells was demonstrated.

Figure 121A shows edited cells that were not lentivirally transduced, transduced with lentivirus encoding Agg520-TCR or transduced with lentivirus encoding Vim418-TCR. Flow cytometry plots analyzing expression of mTCRb in non-transduced edTreg, Agg520 edTreg or Vim418 edTreg cells by gating on LNGFR+Foxp3+ in edited cells. The Agg520 TCR has specificity for an epitope consisting of residues 520-539 of aggrecan. FIG. 121B shows a polyclonal suppression assay using Agg520 or Vim418-specific edTreg or mock cells and Agg520 Teff cells. Aggrecan-specific Teff cells were generated by transducing CD4+ T cells with lentivirus encoding Agg520-TCR and expanded for 15 days. Agg520 Teff cells were incubated without Treg cells or with edTreg cells or mock cells in the presence of anti-CD3/CD28 beads. Figure 121C shows the percent growth inhibition of Agg520 Teff cells by no Treg cells, untransduced (untd) edTreg cells, Agg edTreg cells, Agg mock cells, Vim edTreg cells or Vim mock cells. 2 shows a graph showing the results calculated from the ratio. FIG. 121D shows antigen-specific and bystander suppression assays using Agg520 or Vim418-specific edTreg or mock cells and Agg520 Teff cells. Agg520 Teff cells were cultured without adding Treg cells, or edTreg cells or mock cells and Agg520 Teff cells were co-cultured in the presence of Agg520 peptide or Agg520 peptide+Vim418 peptide and APC. FIG. 121E shows a graph showing the results of calculating the growth inhibition rate of Agg520 Teff cells by no Treg cells, edTreg cells, or mock cells from the growth rates shown in FIG. 121D. Importantly, vimentin-specific edTreg cells were shown to exert a bystander suppressive effect on aggrecan-specific Teff cells.

Antigen-specific suppression and bystander suppression effects on CILP-specific Teff cells
By using CILP-specific edTreg cells, antigen-specific suppression on CILP-specific Teff cells was shown, and by using vimentin-specific edTreg cells, bystander suppression on CILP-specific Teff cells. Effect was shown. Figure 122A shows edited cells not transduced with lentivirus, edited cells transduced with lentivirus encoding CILP297-1-TCR or transduced with lentivirus encoding Vim418-TCR. Flow cytometry plots analyzing mTCRb expression in non-transduced edTreg, CILP297-1 edTreg or Vim418 edTreg cells by gating on LNGFR+Foxp3+ in edited cells. FIG. 122B shows a polyclonal suppression assay using CILP297- or Vim418-specific edTreg or mock cells and CILP297-1 Teff cells. CILP-specific Teff cells were generated by transducing CD4+ T cells with lentivirus encoding CILP297-1-TCR and expanded for 15 days. CILP297-1 Teff cells were incubated without Treg cells or with edTreg cells or mock cells in the presence of anti-CD3/CD28 beads. Figure 122C shows the percent growth inhibition of CILP Teff cells by no Treg cells, untransduced (untd) edTreg cells, CILP edTreg cells, CILP mock cells, Vim edTreg cells, or Vim mock cells. 2 shows a graph showing the results calculated from the ratio. FIG. 122D shows antigen-specific and bystander suppression assays using CILP297 or Vim418-specific edTreg cells and CILP297-1 Teff cells. CILP297-1 Teff cells were cultured without adding Treg cells, or CILP297-1 Teff cells were co-cultured with edTreg cells or mock cells in the presence of CILP297 peptide or CILP297 peptide+Vim418 peptide and APC. FIG. 122E shows a graph showing the results of calculating the growth inhibition rate of CILP Teff cells by no Treg cells, edTreg cells, or mock cells from the growth rate shown in FIG. 122D. Importantly, Vim edTreg cells were shown to exert a bystander suppressive effect on CILP297-1-specific Teff cells.

SLE-specific edTreg cells and their suppressive activity
SLE-specific edTreg cells were generated. A SLE3 TCR previously identified from a systemic lupus erythematosus patient was transduced into CD4 T cells to edit the Foxp3 locus.

Figure 123A shows flow cytometry plots showing expression of mTCRb and LNGFR/Foxp3 at day 7 in edited cells expressing SLE3-TCR. The SLE3-TCR was previously identified from a patient with systemic lupus erythematosus. At day 10, edTreg cells were enriched by expression of LNGFR. LNGFR- cells were used as mock cells for suppression assays. SLE3-TCR had specificity for an epitope consisting of residues 65-80 of SmD1. FIG. 123B shows polyclonal and antigen-specific suppression assays using SLE-specific edTreg cells. SLE-specific Teff cells were generated by transducing CD4+ T cells with lentivirus encoding SLE3-TCR and expanded for 15 days. For polyclonal suppression assays, SLE3 Teff cells were incubated without Treg cells or with SLE3 edTreg cells or mock cells in the presence of anti-CD3/CD28 beads. For antigen-specific suppression assays, SLE3 Teff cells, APCs and SmD1 peptide were co-cultured in the absence of Treg cells or in the presence of SLE3 edTreg cells or mock cells. Edited cells express SLE-TCR and have polyclonal and antigen-specific suppressive activity.

At least the data presented in this example demonstrate that edTreg cells specific for type 1 diabetes, rheumatoid arthritis or SLE antigens can be generated and that these edTreg cells have suppressive activity. , suggested that antigen-specific edTreg cell therapy could be used for a wide range of autoimmune diseases.

Example 13 - Double Editing of Human CD4+ T Cells Double editing human CD4+ T cells to have endogenous TCR knocked out/inactivated, antigen specific and/or drug to generate selectable edTreg cells.

In a single locus-editing homologous recombination repair (HDR)-based double-editing method, a split-type IL-2 CISC system was used to efficiently generate double-edited cells in which the endogenous TCR was knocked out. Selected and concentrated. A problem with the double-editing method is that it is difficult to obtain sufficient numbers of edited cells for therapeutic use. The purpose of this study was to improve the viability of cells in the expansion culture stage. The AAV HDR donor construct targeting the TRAC locus used in this study is shown in Figure 124A. This AAV HDR donor construct was designed to introduce each component of the split-type CISC into the TRAC locus using a single-locus double-editing strategy. The CISC components were split between two constructs and co-expressed with HA-FOXP3 (No.3240) or T1D4 TCR (No.3243). Each repair template was flanked by homologous arms that matched the gRNA targeting the TRAC locus. Only CD4+ T-edited cells that had integrated both expression cassettes were predicted to selectively expand upon exposure to the rapalog.

The timeline of the double editing process of CD4+ T cells with AAV and the expansion culture process in the presence of rapalogs, which are important in this study, are shown in Figure 124B. The expansion protocol was changed from 10 days expansion in the presence of AP21967 (a rapamycin analogue) to 7 days expansion in the presence of AP21967 followed by 3 days recovery in medium containing IL-2. changed. Briefly, using AAV construct No.3240 (MND.HA.FOXP3.FKBP.IL2RG) and AAV construct No.3243 (MND.T1D4.FRB.IL2RB) and human TRAC gRNA_4 (one gene locus double editing), gene editing of human CD4+ T cells was performed. Immediately after electroporation, cells were seeded in medium containing 2.5% FBS (recovery medium) and cultured for about 24 hours, and maintained in medium containing 20% FBS for the remainder of the experiment. FACS analysis was performed on day 3 to measure the editing rate, and the edited cell population was cultured in the presence of IL-2 or rapalogs for an additional 7 days to enrich for FOXP3/T1D4 positive double edited cells. Cells were allowed to recover in media containing IL-2 for 3 days before FACS analysis on day 14.

FOXP3/T1D4 in which expression of the TCR was disrupted by double editing the human TRAC locus in human CD4+ T cells using constructs containing FOXP3 and split CISC and constructs containing T1D4 and split CISC. Double positive cells were obtained. Figure 125A shows the expression of T1D4 and FOXP3 in mock-edited, single-edited and double-edited cells (using 10% by volume of AAV No. 3243 and AAV No. 3240, respectively) 3 days after gene editing. Flow cytometry plots are shown. The virus titer of No.3243 was 4.2×10 ¹¹ and the virus titer of No.3240 was 1.3×10 ¹² . Figure 125B shows flow cytometry plots showing expression of T1D4 and CD4 in mock-edited cells and cells edited with the virus mixture. FIG. 125C shows histograms showing the percentage of double-negative, FOXP3-HA-positive, T1D4-positive, and FOXP3/T1D4 double-positive cells in double-edited cells. FIG. 125D shows histograms comparing CD3 knockout rates in FOXP3/T1D4 double-edited cells and mock-edited cells. FACS analysis showed an initial editing rate of 1.6% in T1D4/FOXP3 double-edited cells compared to 0% in mock-edited cells, indicating knockout of CD3 in double-edited cells. (KO) rate was shown to be 70%.

By treating the double-edited cells with AP21967, we observed a high enrichment of double-edited cells that expressed FOXP3/T1D4 and increased expression of CTLA4. Double editing of the TRAC locus was performed by the method shown in Figures 124A and 124B. Figure 126A Flow cytometry showing viability and expression of T1D4 and FOXP3 after treatment of double-edited cells with 50 ng/mL IL-2 (top panel) or 100 nM rapalog (AP21967; bottom panel) for 7 days. Show the plot. FIG. 126B shows CTLA4 expression in T1D4/FOXP3 double-positive and double-negative cell populations after treatment with 50 ng/mL IL-2 (top panel) or 100 nM rapalog (AP21967; bottom panel) for 7 days. shows flow cytometry plots comparing . FACS analysis 7 days after enrichment showed that the percentage of double-positive cells on day 7 treated with IL-2 was 1.47%, compared to double-positive cells on day 7 treated with AP21967. reached 19.1%, indicating a stable increase in FOXP3/T1D4 double-positive cells over time.

The viability of AP21967-treated cells was lower than that of cells treated with 50 ng/mL IL-2 (AP21967-treated cells had 11% viability compared to 50 ng/mL IL-2). The viability of cells treated with -2 was 95%) (Fig. 126A). To improve cell viability after treatment with AP21967, cells were treated with AP21967 for 7 days and then cultured in medium containing 50 ng/mL IL-2. When AP21967-treated cells were allowed to recover in media containing IL-2, we observed enhanced viability and continued enrichment of dual-edited cells expressing FOXP3/T1D4. Double editing of the TRAC locus was performed by the method shown in Figures 124A and 124B. Cells were analyzed on day 10 after recovery in medium containing IL-2 for 3 days. Figure 127A shows double-edited cells treated with 50 ng/mL IL-2 (top plot) and double-edited cells treated with 100 nM AP21967 and then allowed to recover in medium containing IL-2 (bottom plot). Flow cytometry plots comparing survival (right plots) and expression of T1D4 and FOXP3 (left plots) are shown in . FIG. 127B shows T1D4/FOXP3 double-positive cells treated with 50 ng/mL IL-2 or 100 nM rapalog (AP21967) and allowed to recover in recovery medium containing IL-2 for the last three days. FIG. 10 shows a graph showing fold enrichment over 10 days. Recovery in media containing IL-2 increased overall viability from 11% to 20.7% (Figures 126A and 127A), and the percentage of FOXP3/T1D4 double-positive cells continued to 24.9%. Increased. Overall, the antigen-specific double-positive Treg cell population was enriched approximately 15-fold throughout the study (Fig. 127B), suggesting that this method could improve survival and expansion.

To further characterize the T1D4/FOXP3-expressing cells, we measured the expression of CTLA4, a marker of FOXP3-expressing natural regulatory T cells (nTreg). FIG. 126B shows that CTLA4 expression was increased in T1D4/FOXP3-expressing double-positive cells compared to the double-negative population, consistent with having a Treg-like phenotype.

Furthermore, in this study, we show that the double-editing method can introduce a TCR candidate and a split IL2 CISC cassette, and double-edited cells can be enriched with rapalogs to generate antigen-specific edTreg cells. .

Decoy - Double Editing with CISC Construct (Split DISC Construct)
Rapamycin can be used in clinical studies with CISC-expressing edTreg cells. A 'decoy-CISC (DISC)' construct was tested for efficient enrichment using rapamycin or AP21967. Enrichment and expansion of double-edited T cells was investigated using a split DISC construct. Edited CD4+ T cells were expanded in gREX flasks to assess the feasibility of scaling up the manufacturing process to obtain sufficient numbers of cells for animal studies. Specifically, a split DISC construct was used to assess dual editing of human CD4+ T cells and their enrichment. Briefly described below. An HDR knock-in construct (No. 3280) containing a split IL-2 DISC for selection of double edited cells with rapamycin or rapalog is shown in Figure 128A. To create a split decoy-CISC (split DISC), a free FRB domain to sequester rapamycin in the cytoplasm was added to the MND.mCherry.FKBP.IL2RG construct to give MND.mCherry.FKBP.IL2RG.FRB (No.328) was obtained. Each repair template (No.3280 and No.3207) was flanked by identical homologous arms matched to gRNAs targeting the TRAC locus. It was expected that CD4+ T cells edited by integrating one copy of each construct could be selectively expanded by treatment with rapalogs or rapamycin. Figure 128B shows the timeline of the dual editing steps of CD4+ T cells using AAV No.3280 and AAV No.3207, the expansion steps in the presence of rapalog/rapamycin, and the analysis steps of the enriched cells. Cells were stimulated with CD3/CD28 beads for 3 days prior to gene editing. Immediately after electroporation, cells were seeded in medium containing 2.5% FBS (recovery medium) and cultured for about 24 hours, and maintained in medium containing 20% FBS for the remainder of the experiment. Three days after gene editing, cells were analyzed for GFP and mCherry expression by flow cytometry and cells were expanded in media containing 50 ng/ml human IL-2 or 100 nM rapalog.

By double-editing human CD4+ T cells using a decoy-CISC (split DISC) construct and enriching with AP21967, double-positive cells could be expanded to large numbers. Figure 129A is a flow cytometry plot showing the expression of mCherry and GFP in double-edited cells (AAV donor No. 3280 and AAV donor No. 3207 each added to 10% of medium volume) 4 days after gene editing. indicate. The virus titer of No.3280 was 3.30×10 ¹² and the virus titer of No.3207 was 3.1×10 ¹⁰ . Figure 129B Flow cytometry showing viability (upper panel) and GFP and mCherry expression (lower panel) after seeding 7.6 million edited cells in gREX flasks and expanding in the presence of AP21967 for 7 days. The plot shows a 32-fold increase in double-positive cells. FACS analysis showed that 7 days of expansion in the presence of AP21967 in gREX flasks enriched for mCherry/GFP double-positive cells with an initial editing rate of 4.47%, increasing to 66%. confirmed. These results indicate that treatment with AP21967 for 7 days resulted in a 32-fold increase in double-positive cells, yielding a total of 11.1 million double-positive cells from the 340,000 cells initially seeded in gREX flasks. was shown to be obtained.

A second study was conducted using a protocol substantially similar to the experiment just described. By double-editing human CD4+ T cells using a decoy-CISC (split DISC) construct and enriching with AP21967, double-positive cells could be expanded to large numbers. Figure 130A is a flow cytometry plot showing the expression of mCherry and GFP in double-edited cells (AAV donor No. 3280 and AAV donor No. 3207 each added to medium volume at 10%) 4 days after gene editing. indicate. The virus titer of No.3280 was 3.30×10 ¹² and the virus titer of No.3207 was 3.1×10 ¹⁰ . Figure 130B Flow cytometry showing viability (upper panel) and GFP and mCherry expression (lower panel) after seeding 7.6 million edited cells in gREX flasks and expanding in the presence of AP21967 for 7 days. The plot shows a 32-fold increase in double-positive cells.

Massive expansion of double-edited human CD4+ T cells could be recapitulated using a decoy-CISC (split DISC) construct. Figure 130A shows the times of the double editing step of CD4+ T cells using AAV No.3280 and AAV No.3207, the expansion step in the presence of rapalogs, and the analysis step of the enriched cells, which are important in this study. indicate the line. Cells were stimulated with CD3/CD28 beads for 3 days prior to gene editing. Three days after gene editing, cells were analyzed for GFP and mCherry expression by flow cytometry and cells were expanded for an additional 7 days in medium containing 100 nM rapalog. FIG. 130B shows flow cytometry plots showing expression of mCherry and GFP in double edited cells (AAV No.3280 and AAV No.3207 added at 10% each). The virus titer of No.3280 (MND.mCherry.FKBP.IL2RG.FRB) was 3.30×10 ¹² and that of No.3207 (pAAV.MND.GFP.FRB.IL2RB) was 3.1×10 ¹⁰ . there were.

Expansion of double-edited human CD4+ T cells in the presence of AP21967 using a decoy-CISC (split DISC) construct allowed a 45-fold increase in enriched cells. Double editing of cells was performed by the method shown in Figure 130A. Figure 131 shows flow cytometry plots showing viability and expression of GFP and mCherry after gREX flasks were seeded with edited cells and expanded in the presence of AP21967 for 7 days. The total number of double-positive cells in the gREX flask on day 7 was 9.7 million, an approximately 45-fold increase from the initial seeding of 216,000 double-positive cells.

Importantly, in this study, we were able to increase double-edited cells by at least about 45-fold by double-editing the TRAC locus using a split DISC construct. Expansion at such folds was comparable to expansion with single gene editing using undivided DISC constructs. Therefore, this method can efficiently enrich double-edited cells for in vivo transplantation studies and ultimately for clinical application.

Studies of Antigen-Specific Mouse Treg Cells To assess the functional activity of antigen-specific mouse edTreg cells, an antigen-specific in vitro suppression assay was constructed. Proliferation of BCD2.5 (islet antigen-specific) Teff cells was assessed. BCD2.5 (islet antigen-specific) Teff cells were activated with BDC peptide in the presence or absence of BCD2.5-expressing MND.LNGFR.P2A edTreg cells or purified BCD2.5 TCR-expressing nTreg cells. Briefly described below. Figure 132A shows an in vitro suppression assay using mouse edTreg cells or mouse nTreg cells. Treg cells edited with MND.LNGFR.p2A (No.3261) were enriched using an anti-LNGFR column two days after gene editing and resuspended in RPMI medium containing 10% FBS. nTreg cells (CD4+CD25+), Teff cells (CD4+CD25+) and antigen presenting cells (APC) (CD4+CD25+) were isolated from spleen and lymph node cells of 8-10 week old NOD BDC2.5+ mice by column enrichment. ) was isolated. Enriched 5×10 ⁶ Teff cells were resuspended in 2 ml PBS, labeled by adding Cell Trace Violet and incubating at 37° C. for 15 minutes, washed with medium, resuspended in medium. were added to the suppression assay. In the assay setup, 2.0×10 ⁵ irradiated (2500 rad) APCs were loaded with 0.25 μg/ml BDC peptide and plated in a total volume of 250 μl medium in a U-bottom 96-well tissue culture plate and 0.5 ×10 ⁵ Teff cells and counted BDC2.5+nTreg or BDC2.5+edTreg cells were added. These cells were incubated for 4 days at 37°C in a _CO2 incubator. On day 4, cells were washed twice with PBS, subjected to live/dead, anti-CD4, anti-CD45 and CD25 staining and analyzed by FACS (LSRII) to determine the proliferation of Teff cells. The suppressive effect of Treg cells was examined. FIG. 132B shows representative flow cytometry data showing decreased proliferation of BDC2.5+ Teff cells in the presence of BDC2.5+ edTreg cells. The results of this experiment showed that peptide-activated Teff cells were suppressed in the presence of edTreg cells.

Mouse BDC2.5+ nTreg cells and mouse BDC2.5+ edTreg cells were observed to exhibit suppressive function in vitro. Figure 133 shows Cell Trace Violet-labeled CD4+ T cells in the presence or absence of mock cells, MND.LNGFR.p2A (No.3261)-edited Treg cells or nTreg cells from NOD BDC2.5+ mice. Flow cytometry plots showing . Numbers in each flow cytometry plot indicate the percentage of proliferated and non-proliferated cells. Mouse edTreg cells and mouse nTreg cells exhibited potent suppressive functions in vitro. These data indicated that edTreg cells expressing the LNGFR selectable marker (MND.LNGFR.P2A (No.3261)) exhibited antigen-specific suppressive activity in vitro.

Using methods substantially similar to those described in Examples 6 and 11, the in vivo activity of edTreg cells was examined. Specifically, antigen-specific T-cell function was investigated by comparing nTreg cells with column-enriched edTreg cells in an NSG adoptive transfer model. Mice were injected with antigen-specific (BDC) recombinant BDC2.5+edTreg cells or antigen-specific nTreg cells followed by antigen-specific Teff cells. Diabetes in mice was monitored for up to 49 days. Figure 134 shows the percentage of mice that developed diabetes after administration of the indicated mock-edited cells, MND.LNGFR.P2A-edited cells or nTreg cells plus effector cells from NOD BDC2.5 mice. Show the graph. Column-enriched, antigen-specific MND.LNGFR.P2A edTreg cells completely prevented diabetes in NSG mice and performed similarly to nTreg cells.

In another study, mice were injected with antigen-specific (BDC) recombinant BDC2.5+edTreg cells or antigen-specific nTreg cells followed by antigen-specific Teff cells. Diabetes in mice was monitored for up to 33 days. Figure 135 shows the percentage of mice that developed diabetes following administration of the indicated mock-edited cells, MND.LNGFR.P2A-edited cells or nTreg cells plus effector cells from NOD BDC2.5 mice. Show the graph. Column-enriched, antigen-specific MND.LNGFR.P2A edTreg cells completely prevented diabetes in NSG mice and performed similarly to nTreg cells. Strikingly, column-enriched LNGFR BDC2.5 edTreg cells completely prevented diabetes in NSG mice in two separate experiments, showing comparable function to BDC2.5 nTreg cells (Fig. 134 and Fig. 135).

As used herein, the term "comprising" is synonymous with the terms "including," "containing," or "characterized by," and open It is meant in an end-to-end, inclusive sense, and does not exclude additional elements or steps not described herein.

The foregoing description discloses several methods and materials of the present invention. The methods and materials of the invention may vary, as may manufacturing methods and equipment. Such modifications will be readily apparent to those skilled in the art in light of this disclosure or practice of the invention disclosed herein. This invention, therefore, is not limited to the particular embodiments disclosed herein, but encompasses all modifications and other aspects possible within the true scope and spirit of the invention.

All references, including but not limited to published patent applications, unpublished patent applications, patents and scientific literature, cited herein are hereby incorporated by reference in their entirety and form part of To the extent any document, patent, or patent application incorporated by reference conflicts with the disclosure of this specification, the disclosure herein will control and/or supersede such conflict.

Claims (70)

Antigen-specific CD4+CD25+ artificial immunoregulatory T (airT) cells,
(a) an artificially modified FOXP3 gene that constitutively expresses the forkhead box protein 3/winged helix transcription factor (FOXP3) gene product at an expression level equal to or greater than that of natural regulatory T (Treg) cells; and (b) an airT cell comprising at least one introduced polynucleotide encoding an antigen-specific T cell receptor (TCR) polypeptide. Artificial modification of the forkhead box protein 3/winged helix transcription factor (FOXP3) gene in CD4+CD25- T cells and modification of at least one polynucleotide encoding an antigen-specific T cell receptor (TCR) polypeptide Antigen-specific CD4+CD25+ artificial immunoregulatory T (airT) cells obtained by introduction,
airT cells that constitutively express the FOXP3 gene product at an expression level equal to or higher than that of natural regulatory T (Treg) cells through the artificial modification. The FOXP3 gene is present at a FOXP3 locus containing an intronic T regulatory cell (Treg)-specific demethylated region (TSDR) having multiple cytosine-guanine (CG) dinucleotides, each CG dinucleotide 3. The airT cell of claim 1 or 2, which contains a methylated cytosine (C) nucleotide at a nucleotide position that in the Treg cell of , contains a demethylated C nucleotide. At nucleotide positions containing demethylated C nucleotides in the TSDR of natural Treg cells, at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the C nucleotides are methylated. The airT cell of claim 3, wherein the airT cell is 5. The airT cell of any one of claims 1-4, wherein the FOXP3 gene product is expressed in an expression level sufficient for the airT cell to maintain a CD4+CD25+ phenotype for at least 21 days in vitro. Sufficient expression such that said airT cells maintain a CD4+CD25+ phenotype in vivo for at least 60 days after adoptive transfer of said airT cells into an immunocompatible mammalian host in need of antigen-specific immunosuppression. The airT cell of any one of claims 1-5, wherein said FOXP3 gene product is expressed in an amount. 7. The airT cell of any one of claims 1-6, comprising a phenotype selected from (i) HeliosLo, (ii) CD152+, (iii) CD127- and (iv) ICOS+. The airT cell of any one of claims 1-7, wherein said artificial modification comprises knockout of the native FOXP3 locus. Said artificial modification comprises insertion into the native FOXP3 locus of a nucleic acid molecule comprising a constitutively active promoter, which promotes transcription of the endogenous FOXP3-encoding nucleotide sequence of the FOXP3 locus. The airT cell according to any one of claims 1 to 8, which is arranged in the FOXP3 gene so as to be able to 10. The inserted nucleic acid molecule of claim 9, further comprising a nucleic acid sequence encoding a first CISC component capable of specifically binding to a chemically induced signaling complex (CISC) inducible molecule. air T cells. The introduced polynucleotide encoding said TCR polypeptide comprises a nucleic acid sequence encoding a second CISC component, distinct from said first CISC component, capable of specifically binding said CISC-inducing molecule. 11. The airT cell of claim 10, further comprising. wherein said nucleic acid sequence encoding a first CISC component and said nucleic acid sequence encoding a second CISC component are in said CISC-derived molecule, separate from said first CISC component and said second CISC component 12. The airT cell of claim 11, further comprising a nucleic acid sequence encoding a third CISC component capable of specific binding. Claims 9-, wherein said nucleic acid molecule comprising said constitutively active promoter is inserted downstream of an intronic regulatory T cell (Treg)-specific demethylation region (TSDR) of the native FOXP3 locus. 13. The airT cell according to any one of 12. The airT cell of any one of claims 9-13, wherein said constitutively active promoter is the MND promoter. 4. The artificial modification comprises insertion into the native FOXP3 locus of a nucleic acid molecule comprising an exogenous FOXP3-encoding polynucleotide and a constitutively active promoter operably linked thereto. The airT cell according to any one of 1 to 8. The inserted nucleic acid molecule comprising a polynucleotide encoding exogenous FOXP3 and a constitutively active promoter operably linked thereto is specifically directed to chemically-inducible signaling complex (CISC)-inducible molecules. 16. The airT cell of claim 15, further comprising a nucleic acid sequence encoding a first CISC component capable of binding. The introduced polynucleotide encoding said TCR polypeptide comprises a nucleic acid sequence encoding a second CISC component, distinct from said first CISC component, capable of specifically binding said CISC-inducing molecule. 17. The airT cell of claim 16, further comprising. At least one of the nucleic acid sequence encoding the first CISC component and the nucleic acid sequence encoding the second CISC component is different from the first CISC component and the second CISC component, the CISC 18. The airT cell of claim 17, further comprising a nucleic acid sequence encoding a third CISC component capable of specifically binding to the inducer molecule. The nucleic acid molecule, comprising a polynucleotide encoding exogenous FOXP3 and a constitutively active promoter operably linked thereto, causes an intronic regulatory T cell (Treg)-specific deactivation of the native FOXP3 locus. The airT cell according to any one of claims 15 to 18, which is inserted downstream of the methylated region (TSDR). The airT cell of any one of claims 15-19, wherein said constitutively active promoter is the MND promoter. The artificial modification includes insertion of a nucleic acid molecule comprising an exogenous FOXP3-encoding polynucleotide and a constitutively active promoter operably linked thereto at a chromosomal site other than the native FOXP3 locus. airT cells according to any one of claims 1 to 8, comprising. wherein at least one native T cell receptor (TCR) locus of said airT cells is knocked out or inactivated and said at least one introduced polypolypeptide encoding an antigen-specific T cell receptor (TCR) polypeptide; 22. The airT cell of claim 21, substituted with a nucleotide. 23. The airT cell of claim 22, wherein said at least one native TCR locus that is knocked out is the native TCR α chain (TRAC) locus. The inserted nucleic acid molecule comprising a polynucleotide encoding exogenous FOXP3 and a constitutively active promoter operably linked thereto is specifically directed to chemically-inducible signaling complex (CISC)-inducible molecules. The airT cell of any one of claims 21-23, further comprising a nucleic acid sequence encoding a first CISC component capable of binding. The introduced polynucleotide encoding said TCR polypeptide comprises a nucleic acid sequence encoding a second CISC component, distinct from said first CISC component, capable of specifically binding said CISC-inducing molecule. 25. The airT cell of claim 24, further comprising. At least one of the nucleic acid sequence encoding the first CISC component and the nucleic acid sequence encoding the second CISC component is different from the first CISC component and the second CISC component, the CISC 26. The airT cell of claim 25, further comprising a nucleic acid sequence encoding a third CISC component capable of specifically binding the inducing molecule. The airT cell of any one of claims 21-26, wherein said constitutively active promoter is the MND promoter. said chromosomal site, other than the native FOXP3 locus, into which said nucleic acid molecule comprising an exogenous FOXP3-encoding polynucleotide and a constitutively active promoter operably linked thereto has been inserted; The airT cell of any one of claims 21-27, which is within the α chain (TRAC) locus. wherein at least one native T cell receptor (TCR) locus of said airT cells is knocked out or inactivated and said at least one introduced polypolypeptide encoding an antigen-specific T cell receptor (TCR) polypeptide; The airT cell of any one of claims 1-28, which is substituted with a nucleotide. 30. The airT cell of claim 29, wherein said at least one native TCR locus knocked out is the native TCR alpha chain (TRAC) locus. Antigen-specific CD4+CD25+ artificial immunoregulatory T (airT) cells,
(a) an introduced nucleic acid encoding an exogenous forkhead box protein 3/winged helix transcription factor (FOXP3) gene product that is constitutively expressed at levels equal to or greater than those of natural regulatory T (Treg) cells; and (b) at least one introduced polynucleotide encoding an antigen-specific exogenous T-cell receptor (TCR) polypeptide,
wherein said introduced nucleic acid sequence encoding said exogenous FOXP3 gene product comprises a nucleic acid sequence encoding a first CISC component capable of specifically binding to a chemically-inducible signaling complex (CISC)-inducible molecule; further includes;
A nucleic acid in which the introduced nucleic acid sequence encoding the exogenous TCR gene product encodes a second CISC component, separate from the first CISC component, capable of specifically binding to the CISC-inducing molecule. further containing an array,
air T cells. Antigen-specific CD4+CD25+ artificial immunoregulatory T (airT) cells,
(a) the native FOXP3 locus knocked out by homologous recombination repair (i) contains a constitutively active promoter capable of promoting transcription of the endogenous FOXP3-encoding nucleotide sequence of the native FOXP3 gene; (ii) a nucleic acid molecule comprising a nucleotide sequence encoding an exogenous FOXP3 protein or a functional derivative thereof and a constitutively active promoter operably linked thereto, resulting in a natural (b) an antigen-specific exogenous T cell receptor (TCR) knocked out by homologous recombination repair, which constitutively expresses the FOXP3 gene product at levels equal to or greater than regulatory T (Treg) cells; ) contains a native T-cell receptor alpha chain (TRAC) locus into which at least one introduced polynucleotide encoding a polypeptide has been inserted;
said inserted nucleic acid molecule comprising said constitutively active promoter, or said comprising a nucleotide sequence encoding an exogenous FOXP3 protein or a functional derivative thereof and a constitutively active promoter operably linked thereto; the inserted nucleic acid molecule further comprising a nucleic acid sequence encoding a first CISC component capable of specifically binding to a chemically-inducible signaling complex (CISC)-inducible molecule;
A nucleic acid in which the introduced nucleic acid sequence encoding said exogenous TCR polypeptide encodes a second CISC component, distinct from the first CISC component, capable of specifically binding to said CISC-derived molecule further containing an array,
air T cells. At least one of the nucleic acid sequence encoding the first CISC component and the nucleic acid sequence encoding the second CISC component is different from the first CISC component and the second CISC component, the CISC 33. The airT cell of claim 31 or 32, further comprising a nucleic acid sequence encoding a third CISC component capable of specifically binding the inducing molecule. The airT cell comprises at least an introduced first polynucleotide encoding an antigen-specific TCR polypeptide and a second introduced polynucleotide encoding an antigen-specific TCR polypeptide, wherein the first polynucleotide encodes a TCR Vα polypeptide, the second polynucleotide encodes a TCR Vβ polypeptide, and the Vα polypeptide and the Vβ polypeptide are a functional TCR capable of specifically recognizing an antigen The airT cell according to any one of claims 1 to 33, which constitutes a expressing an antigen-specific T-cell receptor (TCR) comprising an antigen-specific TCR polypeptide encoded by at least one introduced polynucleotide encoding said TCR polypeptide and specifically recognized by said TCR polypeptide airT cells according to any one of claims 1 to 34, which are capable of inducing immunosuppression in an antigen-specific manner in response to HLA-restricted stimulation by an antigen that is administered to the airT cell. The immunosuppression induced in an antigen-specific manner is
(i) activation and/or proliferation of effector T cells that recognize an antigen specifically recognized by said airT cell TCR comprising said TCR polypeptide encoded by said at least one introduced polynucleotide; Suppression,
(ii) expression of an inflammatory cytokine or inflammatory mediator by effector T cells that recognize an antigen specifically recognized by said airT cell TCR comprising said TCR polypeptide encoded by said at least one introduced polynucleotide; suppression of
(iii) production of one or more immunosuppressive cytokines, perforin/granzymes or anti-inflammatory products by said airT cells, or against indoleamine-2,3-dioxygenase (IDO), IL2 or adenosine in said airT cells induction of at least one of competition, tryptophan catabolism, and expression of an inhibitory receptor; and (iv) specificity by said airT cell TCR comprising said TCR polypeptide encoded by said at least one introduced polynucleotide. 36. The airT cell of claim 35, comprising one or more of suppressing one or both of activation and proliferation of effector T cells that do not recognize a functionally recognized antigen. The airT cell of any one of claims 1-36, wherein said TCR specifically recognizes an antigen associated with the pathogenesis of an autoimmune, allergic or inflammatory disease. (i) the autoimmune disease is type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, Crohn's disease, bullous pemphigoid, pemphigus vulgaris, autoimmune hepatitis, psoriasis , Sjögren's syndrome and celiac disease;
(ii) said allergic disease is selected from allergic asthma, pollen allergy, food allergy, drug hypersensitivity and contact dermatitis;
(iii) said inflammatory disease is selected from islet cell transplantation, asthma, hepatitis, inflammatory bowel disease (IBD), ulcerative colitis, graft versus host disease (GVHD), transplant tolerance induction, transplant rejection and sepsis to be
38. The airT cell of claim 37. (i) the antigen associated with the pathogenesis of said autoimmune disease is selected from the autoantigens described in any of Figures 141-144;
(ii) the antigen associated with the etiology of the allergic disease is selected from the allergic antigens described in any one of Figures 141-144;
(iii) the antigen associated with the pathogenesis of said inflammatory disease is selected from inflammation-associated antigens according to any of Figures 141-144;
The airT cell of claim 38. 35 or less, 34 or less, 33 or less, 32 or less, 31 or less, 30 or less, 29 or less amino acid sequences selected from the antigenic polypeptide sequences set forth in any of Figures 141 to 144; 28 or less, 27 or less, 26 or less, 25 or less, 24 or less, 23 or less, 22 or less, 21 or less, 20 or less, 19 or less, 18 or less, 17 or less, 16 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less or 7 or less contiguous amino acids. at least one introduced polynucleotide sequence encoding a TCR polypeptide that specifically binds to an HLA restriction, or at least one encoding a TCR polypeptide encoded by a nucleotide sequence set forth in any of Figures 139-140 40. The airT cell of any one of claims 1-39, comprising two introduced polynucleotide sequences. 35 or less, 34 or less, 33 or less, 32 or less, 31 or less, 30 or less, 29 or less amino acid sequences selected from the antigenic polypeptide sequences set forth in any of Figures 141 to 144; 28 or less, 27 or less, 26 or less, 25 or less, 24 or less, 23 or less, 22 or less, 21 or less, 20 or less, 19 or less, 18 or less, 17 or less, 16 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less or 7 or less contiguous amino acids. an introduced first polynucleotide sequence encoding a TCR Vα polypeptide of a TCR that specifically binds to an HLA-restricted TCR and an introduced second polynucleotide sequence encoding a TCR Vβ polypeptide of said TCR; or FIG. an introduced first polynucleotide sequence encoding a TCR Vα polypeptide of a TCR comprising any of the TCRα polypeptide sequences according to any of 136-140 and an introduced polynucleotide sequence encoding a TCR Vβ polypeptide of said TCR a second polynucleotide sequence; or an introduced first polynucleotide sequence encoding a TCR TCR Vα polypeptide encoded by a nucleotide sequence set forth in any of Figures 139-140 and a TCR TCR Vβ polypeptide of said TCR; 41. The airT cell of claim 40, comprising at least an introduced second polynucleotide sequence encoding a. the introduced first polynucleotide sequence encoding a TCR Vα polypeptide of a TCR that specifically binds to an antigenic polypeptide in a human HLA-restricted manner and an introduced first polynucleotide sequence encoding a TCR Vβ polypeptide of the TCR wherein said TCR Vα and Vβ polypeptides are derived from the paired TCR Vα and TCR Vβ polypeptide sequences of any of FIGS. 41. The airT cell of claim 40, comprising selected paired sequences. biological Treg activity induced in said airT cells in response to MHC-restricted stimulation by an antigen recognized by a TCR polypeptide encoded by said at least one introduced polynucleotide is MHC-restricted by the same antigen; is enhanced over the biological Treg activity of the airT cells as a non-stimulated control,
said biological Treg activity is
(i) activation and/or proliferation of effector T cells that recognize an antigen specifically recognized by said airT cell TCR comprising said TCR polypeptide encoded by said at least one introduced polynucleotide; Suppression,
(ii) expression of an inflammatory cytokine or inflammatory mediator by effector T cells that recognize an antigen specifically recognized by said airT cell TCR comprising said TCR polypeptide encoded by said at least one introduced polynucleotide; suppression of
(iii) production of one or more immunosuppressive cytokines, perforin/granzymes or anti-inflammatory products by said airT cells, or against indoleamine-2,3-dioxygenase (IDO), IL2 or adenosine in said airT cells induction of at least one of competition, tryptophan catabolism, and expression of an inhibitory receptor; and (iv) specificity by said airT cell TCR comprising said TCR polypeptide encoded by said at least one introduced polynucleotide. airT cells according to any one of claims 1 to 42, comprising one or more of suppressing one or both of activation and proliferation of effector T cells that do not recognize the antigens that are systematically recognized. (1) the antigen associated with the etiology of an autoimmune disease is an IGRP peptide (residues 241-270), the autoimmune disease is type 1 diabetes, and the TCR is HLA DRB1*0404-restricted; or (2) the antigen associated with the pathogenesis of an autoimmune disease is the IGRP peptide (residues 305-324). 37, wherein the autoimmune disease is type 1 diabetes, and the TCR is TCR T1D5, which recognizes the IGRP peptide (residues 305-324) in an HLA DRB1*0404-restricted manner. airT cells as described in . 44. The airT cell of any one of claims 1-43 for use in the treatment, suppression or alleviation of an autoimmune, allergic or inflammatory disease, wherein said autoimmune disease is type 1 diabetes , multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, Crohn's disease, bullous pemphigoid, pemphigus vulgaris and autoimmune hepatitis, and said allergic disease is an allergic disease selected from allergic asthma, pollen allergy, food allergy, drug hypersensitivity and contact dermatitis, and said inflammatory disease is pancreatic islet cell transplantation, asthma, hepatitis, inflammatory bowel disease (IBD) , ulcerative colitis, graft-versus-host disease (GVHD), transplant tolerance induction, transplant rejection and sepsis, etc., airT cells. 46. The method of claim 45, wherein the airT cell TCR polypeptide binds an antigen associated with a disorder selected from type 1 diabetes, multiple sclerosis, myocarditis, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). airT cells as indicated. 47. The antigen of claim 46, wherein the antigen is selected from the group consisting of vimentin, aggrecan, cartilage intermediate layer protein (CILP), preproinsulin, IGRP (islet-specific glucose-6-phosphatase catalytic subunit-related protein) and enolase. airT cells. 48. The airT cell of claim 46 or 47, wherein said antigen comprises an epitope selected from the group consisting of Enol326, CILP297-1, Vim418, Agg520 and SLE3. The airT cell of any one of claims 46-48, wherein said antigen comprises an epitope having an amino acid sequence set forth in any of SEQ ID NOs: 1363-1376 and 1408-1415. wherein said TCR polypeptide is
50. Any of claims 45-49, comprising a CD3α polypeptide having an amino acid sequence set forth in any of SEQ ID NOs: 1377-1390; and/or a CD3β polypeptide having an amino acid sequence set forth in any of SEQ ID NOs: 1377-1390. or the airT cell according to 1. A pharmaceutical composition comprising the airT cells according to any one of claims 1 to 50 and a pharmaceutically acceptable excipient. Use of airT cells according to any one of claims 1-50 as a medicament. A method for producing antigen-specific artificial immunoregulatory T (airT) cells, comprising:
(a) under conditions and for a time sufficient to knock out the native FOXP3 locus of CD4+ T cells and insert all or part of the FOXP3 locus donor template nucleic acid,
(1) a FOXP3 guide RNA (gRNA) comprising a spacer sequence complementary to a sequence within the native forkhead box protein 3/winged helix transcription factor (FOXP3) gene of CD4+ T cells, or encoding said FOXP3 gRNA; nucleic acids;
(2) a DNA endonuclease capable of forming a complex with the FOXP3 gRNA of (1), or a nucleic acid encoding said DNA endonuclease; and (3) (i) a nucleotide encoding endogenous FOXP3 of said FOXP3 gene. A nucleic acid molecule comprising a constitutively active promoter capable of promoting transcription of a sequence and (ii) a nucleotide sequence encoding a FOXP3 protein or functional derivative thereof and a constitutively active promoter operably linked thereto. and (b) simultaneously with (a) or sequentially with (a) in any order, an antigen-specific A method comprising transducing said CD4+ T cells with at least one polynucleotide encoding a T cell receptor (TCR) polypeptide. step (b)
(i) transducing said CD4+ T cells with at least one retroviral vector comprising a polynucleotide encoding said antigen-specific T cell receptor (TCR) polypeptide; and (ii) said CD4+ T cells. under conditions and for a period of time sufficient to knock out the native T-cell receptor alpha chain (TRAC) locus and insert all or part of the TRAC locus donor template nucleic acid,
(1) a TRAC guide RNA (gRNA) comprising a spacer sequence complementary to a sequence within the natural TRAC locus of said CD4+ T cell, or a nucleic acid encoding said TRAC gRNA;
(2) a DNA endonuclease capable of forming a complex with the TRAC gRNA of (1), or a nucleic acid encoding the DNA endonuclease; and (3) the antigen-specific T-cell receptor (TCR) polypeptide 54. The method of claim 53, selected from introducing into said CD4+ T cells a TRAC locus donor template comprising at least one encoding polynucleotide. A method for producing antigen-specific artificial immunoregulatory T (airT) cells, comprising:
(a) conditions sufficient to knock out the native T cell receptor alpha chain (TRAC) locus of CD4+ T cells and insert all or part of the first TRAC locus donor template nucleic acid and in time,
(1) a first TRAC guide RNA (gRNA) comprising a first spacer sequence complementary to a first sequence in the native TRAC locus of CD4+ T cells, or encoding said first TRAC gRNA; nucleic acids;
(2) a first DNA endonuclease capable of forming a complex with the first TRAC gRNA of (1), or a nucleic acid encoding said first DNA endonuclease; and (3) (i) FOXP3 protein or a nucleic acid molecule comprising a nucleotide sequence encoding a functional derivative thereof and (ii) a nucleic acid molecule comprising a nucleotide sequence encoding a FOXP3 protein or a functional derivative thereof and a constitutively active promoter operably linked thereto. and (b) simultaneously with (a) or sequentially with (a) in any order, said CD4+ T cells under conditions and for a time sufficient to knock out the native T-cell receptor alpha chain (TRAC) locus and insert a second TRAC locus donor template nucleic acid in whole or in part,
(1) a second TRAC guide RNA (gRNA) comprising a second spacer sequence complementary to a second sequence in the TRAC gene, different from the first spacer sequence, or the second TRAC gRNA; encoding nucleic acid;
(2) selected from the same DNA endonuclease as the first DNA endonuclease and a DNA endonuclease different from the first DNA endonuclease, capable of forming a complex with the second TRAC gRNA of (1); or a nucleic acid encoding said second DNA endonuclease; and (3) at least one polynucleotide encoding an antigen-specific T-cell receptor (TCR) polypeptide. A method comprising introducing a TRAC locus donor template into said CD4+ T cells. A method for producing antigen-specific artificial immunoregulatory T (airT) cells, comprising:
Sufficient conditions and time to knock out the native T cell receptor alpha chain (TRAC) locus of CD4+ T cells by homologous recombination repair and insert all or part of the TRAC locus donor template. in
(1) a TRAC guide RNA (gRNA) comprising a spacer sequence complementary to a sequence within the natural TRAC locus of CD4+ T cells, or a nucleic acid encoding said TRAC gRNA;
(2) a DNA endonuclease capable of forming a complex with the TRAC gRNA of (1), or a nucleic acid encoding said DNA endonuclease; and (3) encoding an antigen-specific T-cell receptor (TCR) polypeptide. introducing into a CD4+ T cell a TRAC locus donor template comprising at least one polynucleotide that: One of the donor templates to be inserted (the first donor template) carries a nucleic acid sequence encoding a first CISC component capable of specifically binding to a chemically-inducible signaling complex (CISC)-inducible molecule. further includes
The other of the donor templates to be inserted (the second donor template) encodes a second CISC component, different from the first CISC component, capable of specifically binding to the CISC-inducing molecule. 57. The method of any one of claims 53-56, further comprising a nucleic acid sequence that At least one of the inserted first donor template and the second donor template can specifically bind to the CISC-derived molecule, which is separate from the first CISC component and the second CISC component. 58. The method of claim 57, further comprising a nucleic acid sequence encoding a third CISC component capable of. (a) said DNA endonuclease is selected from CRISPR/Cas, TALENs, meganucleases, megaTALs and zinc finger nucleases;
(b) said constitutively active promoter is MND and said insertion is by a mechanism selected from homologous recombination repair and non-homologous end joining;
(d) the first CISC component and the second CISC component are mutually exclusive selected from IL2RB and IL2RG;
(e) the third CISC component is FKBP; and (f) the CISC-derived molecule is rapamycin. described method. A method for producing an antigen-specific artificial immunoregulatory T (airT) cell according to any one of claims 1-50, wherein the method according to any one of claims 53-59 is carried out method involving A method of treating or ameliorating a subject having a disease requiring antigen-specific immunosuppression, comprising at least one T-cell receptor (TCR) that specifically recognizes an antigen requiring antigen-specific immunosuppression. 44. A method comprising administering to said subject a therapeutically effective amount of the plurality of artificial immunoregulatory T (airT) cells of claim 43 that express ). 62. The method of claim 61, wherein said disease requiring antigen-specific immunosuppression is an autoimmune disease, an allergic disease or an inflammatory disease. (i) the autoimmune disease is selected from type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, Crohn's disease, bullous pemphigoid, pemphigus vulgaris and autoimmune hepatitis be;
(ii) said allergic disease is selected from allergic asthma, pollen allergy, food allergy, drug hypersensitivity and contact dermatitis;
(iii) said inflammatory disease is selected from islet cell transplantation, asthma, hepatitis, inflammatory bowel disease (IBD), ulcerative colitis, graft versus host disease (GVHD), transplant tolerance induction, transplant rejection and sepsis to be
63. The method of claim 62. (i) the antigen associated with the pathogenesis of said autoimmune disease is selected from the autoantigens described in any of Figures 141-144;
(ii) the antigen associated with the etiology of the allergic disease is selected from allergic antigens according to any one of Figures 141-143;
(iii) the antigen associated with the pathogenesis of said inflammatory disease is selected from inflammation-associated antigens according to any of Figures 141-144;
64. The method of claim 63. 51. A method of treating or ameliorating a subject having a disorder comprising administering to said subject the artificial immunoregulatory T (airT) cells of any one of claims 1-50. said disorder is type 1 diabetes, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, rheumatoid arthritis (RA), Crohn's disease, bullous pemphigoid, pemphigus vulgaris, autoimmune hepatitis; allergic diseases such as allergic diseases selected from allergic asthma, pollen allergy, food allergy, drug hypersensitivity and contact dermatitis; and pancreatic islet cell transplantation, asthma, hepatitis, inflammatory bowel disease (IBD), ulcerative colitis, 66. The method of claim 65, selected from the group consisting of inflammatory diseases, such as inflammatory diseases selected from graft-versus-host disease (GVHD), transplant tolerance induction, transplant rejection and sepsis. 66, wherein said airT cell TCR polypeptide binds an antigen associated with a disorder selected from type 1 diabetes, multiple sclerosis, myocarditis, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), or 66. The method according to 66. said TCR polypeptide binds to an antigen selected from the group consisting of vimentin, aggrecan, cartilage intermediate layer protein (CILP), preproinsulin, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) and enolase , the method of any one of claims 65-67. 69. The method of any one of claims 65-68, wherein said TCR polypeptide binds an antigen comprising an epitope having an amino acid sequence set forth in any of SEQ ID NOs: 1363-1376 and 1408-1415. wherein said TCR polypeptide is
69. Any of claims 65-69, comprising a CD3α polypeptide having an amino acid sequence set forth in any of SEQ ID NOs: 1377-1390; and/or a CD3β polypeptide having an amino acid sequence set forth in any of SEQ ID NOs: 1377-1390. or the method according to item 1.

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